WO2004069191A2 - Couplage fonctionnel de t1rs et de t2rs par des proteines gi, et dosages a base de cellules pour l'identification de modulateurs de t1r et t2r - Google Patents

Couplage fonctionnel de t1rs et de t2rs par des proteines gi, et dosages a base de cellules pour l'identification de modulateurs de t1r et t2r Download PDF

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WO2004069191A2
WO2004069191A2 PCT/US2004/002987 US2004002987W WO2004069191A2 WO 2004069191 A2 WO2004069191 A2 WO 2004069191A2 US 2004002987 W US2004002987 W US 2004002987W WO 2004069191 A2 WO2004069191 A2 WO 2004069191A2
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compound
cells
camp
tir
activity
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WO2004069191A3 (fr
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Guy Servant
Mark Ozeck
Paul Brust
Hong Xu
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Senomyx Inc.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to novel methods and materials for the identification of modulators, e.g., enhancers, agonists and antagonists of G protein-coupled receptors (GPCRs) involved in taste, i.e., TlRs and T2Rs.
  • GPCRs G protein-coupled receptors
  • modulators may be used as flavor- affecting additives, e.g., in foods, beverages and medicines for human or animal consumption.
  • the present invention provides MAP Kinase, cAMP and adenylyl cyclase cell-based assays for the identification of modulators of GPCRs involved in taste modulation, i.e.,
  • T2Rs and TlRs preferably human TlRs and T2Rs.
  • the invention provides cell based assays, e.g., MAP Kinase, cAMP accumulation and adenylyl cyclase cell-based assays that rely on the discovery that G proteins other than gustducin and promiscuous and pernicious, G proteins such as G ⁇ is, i.e., Gi proteins functionally couple to TlRs and T2Rs and use G ⁇ i to transmit signals to downstream effectors.
  • G proteins other than gustducin and promiscuous and pernicious G proteins
  • G proteins such as G ⁇ is, i.e., Gi proteins functionally couple to TlRs and T2Rs and use G ⁇ i to transmit signals to downstream effectors.
  • G proteins heterotrimeric GTP binding proteins
  • GPCRs The family of receptors that transmit signals through the activation of heterotrimeric GTP binding proteins (G proteins) constitutes the largest group of cell surface proteins involved in signal transduction. These receptors participate in a broad range of important biological functions and are implicated in a number of disease states. More than half of all drugs currently available influence GPCRs. These receptors affect the generation of small molecules that act as intracellular mediators or second messengers, and can regulate a highly interconnected network of biochemical routes controlling the activity of several members of the mitogen-activated protein kinase (MAPK) superfamily.
  • MAPK mitogen-activated protein kinase
  • MNK mitogen-activated protein kinase
  • the activation of members of the mitogen-activated protein kinase (MAPK) family represents one of one of the major mechanisms used by eukaryotic cells to transduce extracellular signals into cellular responses (J. Blenis, Proc. Natl. Acad. Sci., USA 90:5889 (1993) (1); Blumer et al., TIBS 19:236 (1994) (2); Cano et al., TIBS 20:117 (1995) (3); Seger et al., FASEB J. 9:726 (1995) (4): R.J. Davis, TIBS 19:470 (1994) (5)).
  • the MAPK superfamily consists of the p42 (ERK2)/p44 (ERKl) MAPKs and the stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38 MAPK. (Robinson and Dickenson, Eur. J. Pharmacol. 413(2-3): 151-61 (2001)(6)).
  • Mitogen-activated protein kinase (also called extracellular signal-regulated kinases or ERKs) are rapidly activated in response to ligand binding by both growth factor receptors that function as tyrosine kinases (such as the epidermal growth factor (EGF) receptor) and receptors that are complexed with heterodimeric guanine nucleotide binding proteins (G proteins) such as the thrombin receptor.
  • G proteins heterodimeric guanine nucleotide binding proteins
  • receptors like the T cell receptor (TCR) and B cell receptor (BCR) are non-covalently associated with src family tyrosine kinases which activate MAPK pathways.
  • MAPKs can also regulate MAPK pathways.
  • the MAPKs appear to integrate multiple intracellular signals transmitted by various second messengers.
  • MAPKs phosphorylate and regulate the activity of enzymes and transcription factors including the EGF receptor, Rsk 90, phospholipase A , c- Myc, c-Jun and EIK-1/TCF.
  • EGF receptor the EGF receptor
  • Rsk 90 the EGF receptor
  • phospholipase A phospholipase A
  • c-Myc c-Myc
  • c-Jun EIK-1/TCF.
  • T2Rs functional receptors
  • TlRs TlRs
  • T1R2 and T1R3 TlRs
  • T2Rs which have been cloned from different mammals including rats, mice and humans (12) (Adler et al., Cell 100(6): 611-8 (2000)). T2Rs comprise a novel family of human and rodent G protein-coupled receptors that are expressed in subsets of taste receptor cells of the tongue and palate epithelia. These taste receptors are organized in clusters in taste cells and are genetically linked to loci that influence bitter taste. The fact that T2Rs modulate bitter taste has been demonstrated in cell-based assays.
  • mT2R-5, hT2R-4 and mT2R-8 have been shown to be activated by bitter molecules in in vitro gustducin assays, providing experimental proof that T2Rs function as bitter taste receptors. (80) (Chandrasheker et al., Cell 100(6): 703 (2000)).
  • the present assignee has filed a number of patent applications relating to various T2R genes and the corresponding polypeptides and their use in assays, preferably high-throughput cell-based assays for identifying compounds that modulate the activity of T2Rs.
  • These Senomyx applications i.e., U.S. Serial No. 09/825,882, filed on April 5, 2001, U.S. Serial No. 191,058 filed July 10, 2002 and U.S. Provisional Application Serial No. 60/398,727, filed on July 29, 2002 all incorporated by reference in their entireties herein.
  • the present assignee has exclusively licensed patent applications relating to T2R genes which were filed by the University of California i.e., U.S. Serial No.
  • TlRl The three TIR gene members TlRl, T1R2 and T1R3 form functional heterodimers that specifically recognize sweeteners and amino acids (14-16) (Li et al., Proc. Natl Acad Sci., USA 99(7):4692-6 (2002); Nelson et al., Nature
  • T1R1/T1R3 combination recognizes several L-amino acids and monosodium glutamate (MSG), respectively (14, 15) (Li et al., Proc. Natl Acad Sci., USA 99(7):4692-6 (2002);, Nelson et al., Nature (6877):199-202 (2002)). These results, demonstrate that TlRs are involved in sweet and umami taste.
  • the co-expression of TlRl and T1R3 in recombinant host cells results in a hetero-oligomeric taste receptor that responds to umami taste stimuli.
  • Umami taste stimuli include by way of example monosodium glutamate and other molecules that elicit a "savory" taste sensation.
  • the co- expression of T1R2 and T1R3 in recombinant host cells results in a hetero- oligomeric sweet taste receptor that responds to both naturally occurring and artificial sweeteners.
  • TlR DNAs and the corresponding polypeptides have significant application in cell and other assays, preferably high throughput assays, for identifying molecules that modulate TIR taste receptors; particularly the T1R2/T1R3 receptor (sweet receptor) and the T1R1/T1R3 receptor (umami receptor).
  • TlR modulators can be used as flavor- affecting additives in foods, beverages and medicines.
  • G proteins other than G i5 and gustducin (Gi proteins) which functionally couple to GPCRs involved in taste, i.e., TlRs and T2Rs.
  • MAPK activation will be measured using polyclonal or monoclonal antibodies that specifically recognize activated forms of MAPK, e.g., antibodies that specifically bind p42/p44 MAPK or p38 MAPK or will be measured using proximity assays (e.g., AlphaScreenTM from Packard or High Content Screening Systems (e.g., ERK, MAPK Activation HitKitTM from Cellomics).
  • proximity assays e.g., AlphaScreenTM from Packard or High Content Screening Systems (e.g., ERK, MAPK Activation HitKitTM from Cellomics).
  • cAMP levels are measured by immunoassay methods, optionally after cAMP accumulation is induced by the use of a compound such as forskolin.
  • T2R or TIR agonists or antagonists identified using the subject cell-based assays that monitor the effects of a compound on G ⁇ i mediated signaling pathways, e.g., cAMP, MAPK and adenylyl cyclase assays.
  • T2R or TIR modulatory compounds as flavor-affecting additives, e.g., in foods, beverages and medicaments for human or animal consumption.
  • compositions containing T2R or TlR modulatory compounds identified using the subject cell- based MAPK and cAMP assays are yet another object of the invention.
  • a mammalian cell line such as HEK-293
  • Figure 1 contains the results of an experiment showing that mT2R5 couples to activation of ERK1/2 MAPK.
  • Panel A contains results of an experiment wherein mT2R5-expressing HEK293 cells were incubated with buffer alone (HBSS), 100 ng/mL EGF, 40 ⁇ M cycloheximide, 250 ⁇ M quinine, 2 mM denatonium, 2 mM saccharin, 100 mM sucrose, or 5 mM MSG/lmM IMP in HBSS for 5 minutes at 37°C.
  • buffer alone 100 ng/mL EGF
  • 40 ⁇ M cycloheximide 250 ⁇ M quinine
  • 2 mM denatonium 2 mM saccharin
  • 100 mM sucrose or 5 mM MSG/lmM IMP in HBSS for 5 minutes at 37°C.
  • Cell lysate proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes and then blotted using antibodies directed against phosphorylated ERK1/2 MAPK.
  • PTX-treated cells were incubated with 100 ng/mL PTX overnight prior to experiment.
  • Panel B contains an experiment that measured the course of cycloheximide-induced ERKl/2 phosphorylation in mT2R5-expressing cells.
  • Panel C contains an experiment wherein HEK293 cells transiently expressing rT2R9 were treated as described in Panel A.
  • Panel D contains an experiment showing the effect of increasing concentrations of cycloheximide on ERKl/2 activation.
  • mT2R5-expressing HEK293 cells were incubated with cycloheximide diluted in HBSS (0.1 to 100 NM) for 5 minutes at 37°C. Cell lysate proteins were analyzed as described in Panel A. Bands (inset) were quantified and data were normalized to maximal stimulation of phospho- ERKl/2 MAPK (at 100 ⁇ M cycloheximide)
  • Panel E contains an experiment wherein naive HEK293 cells were treated as described in Panel A.
  • the results in Panels A, D and E are representative of at least 3 independent experiments.
  • the results in Panels B and C are representative of two independent experiments.
  • Figure 2 contains experiments which demonstrate that hTlR2/R3 and hTlRl/R3 couple to activation of ERKl/2 MAPK.
  • Panel A contains an experiment wherein hTlR2 R3-expressing HEK293/G15 cells incubated with buffer alone (D-PBS), 100 ng/mL EGF, 40 ⁇ M cycloheximide, 250 ⁇ M quinine, 2 mM denatonium, 2 mM saccharin, 100 mM sucrose, 5mM MSG/7mM IMP, 4mM D-tryptophane and lOniM cyclamate in D-PBS for 5 minutes at 37°C.
  • Cell lysate proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes and then blotted using antibodies directed against phosphorylated ERKl/2 MAPK.
  • PTX-treated cells were incubated with 100 ng/mL PTX overnight prior to the experiment.
  • Panel B contains an experiment wherein hTlRl hTlR3- expressing HEK293/G15 cells were treated with mifepristone to induce receptor expression (described infra) 48 hours later, cells were incubated with buffer alone (D-PBS), 100 ng/mL EGF, 40 ⁇ M cycloheximide, 250 ⁇ M quinine, 2 mM denatonium, 2mM saccharin, lOOmM sucrose and 5mM MSG/lmM IMP in D- PBS for 5 minutes at 37°C. Cell lysate proteins were analyzed as described in Panel A.
  • Panel C contains an experiment wherein naive HEK293/G15 cells were treated as described in Panel B. (Results therein are representative of at least 3 independent experiments).
  • Figure 3 contains experiments showing the effects of increasing concentrations of sweeteners and MSG on ERKl/2 activation.
  • Panels A and B contain experiments wherein hTlR2/hTlR3-expressing HEK293/G ⁇ s cells were incubated with increasing concentrations of either saccharin (Panel A) (0.078 to 10 mM) or sucrose (Panel B) (3.13 to 400 mM) for 5 minutes at 37°C.
  • Cell lysate proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes and then blotted using antibodies directed against phosphorylated ERKl/2 MAPK.
  • Bands (insets) were quantified and data were normalized to maximal stimulation of phospho-ERKl/2 MAPK (at 10 mM and 400 mM saccharin and sucrose respectively).
  • Panel C contains an experiment wherein hTlRl/hTlR3- expressing HEK293/G15 cells were induced for receptor expression as described in the methods section (infra). Cells were then incubated with increasing concentrations of MSG (0.03 to 60 mM) in the absence or presence of 10 mM IMP for 5 minutes at 37°C. Cell lysate proteins were then analyzed as described in A. Bands (inset) were quantified and data were normalized to maximal stimulation of phospho-ERKl/2 MAPK (at 10 mM and 60 mM MSG). These results are representative of at least three independent experiments.
  • Figure 4 contains experiments which show that cycloheximide inhibits cAMP accumulation in mT2R5-expressing cells.
  • Panel A contains an experiment wherein mT2R5- expressing HEK293 and naive HEK293 cells were incubated with 0.7 ⁇ M forskolin and 50 ⁇ M rolipram in the absence and presence of 40 ⁇ M cycloheximide in HBSS for 15 minutes at 37°C.
  • cAMP levels were determined as described in the methods section infra.
  • cAMP content of mT2R5-expressing cells stimulated with buffer (0.525% DMSO in HBSS) was 5 pmol/well.
  • cAMP content of mT2R5-expressing cells stimulated with forskolin was 73 pmol/well.
  • Cells were also treated with lOOng/ml PTX for 4 hours at 37°C and then stimulated as described above.
  • the cAMP content of mT2R5-expressing cells stimulated with buffer (0.525% DMSO in HBSS) was 4 pmol/well and cAMP content of mT2R5-expressing cells stimulated with forskolin was 80 pmol well.
  • Panel B contains an experiment comparing the effect of increasing concentrations of cycloheximide on forskolin-induced cAMP accumulation.
  • Panel A contains an experiment wherein hTlR2/hTlR3-expressing HEK293/G15 cells were incubated with 5 ⁇ M forskolin and 50 ⁇ M rolipram in the absence and presence of either 200mM fructose, 200mM sucrose, ImM aspartame, 3mM cyclamate, 2mM saccharin or 50 ⁇ M monellin in D-PBS for 15 minutes at 37°C and cAMP levels were determined as described in the methods section.
  • cAMP content of cells stimulated with buffer 0.525% DMSO in D-PBS
  • cAMP content of mT2R5-expressing cells stimulated with forskolin was 23 pmol/well.
  • Panel B shows naive HEK293/G15 cells that were treated as in Panel A.
  • Cells stimulated with buffer (0.525% DMSO in D-PBS) was 4 pmol/well and cAMP content of cells stimulated with forskolin was 90 pmol/well:
  • Panel C contains an experiment comparing the effects of increasing concentrations of cyclamate on forskolin-induced cAMP accumulation.
  • cAMP content of cells stimulated with forskolin alone was 11 pmol/well.
  • Panel D contains an experiment comparing the effectss of increasing concentration of aspartame on forskolin- induced cAMP accumulation. Cells were incubated with of 5 ⁇ M forskolin and 50 ⁇ M rolipram in the absence or presence of increasing concentrations of aspartame (0.03 to 4 mM). cAMP content of cells stimulated with forskolin alone was 14 pmol/well.
  • Panel E contains an experiment comparing the effects of increasing concentration of saccharin on forskolin-induced cAMP accumulation.
  • Cells were incubated with of 5 ⁇ M forskolin and 50 ⁇ M rolipram in the absence or presence of increasing concentrations of saccharin (0.008 to 1 mM).
  • cAMP content of cells stimulated with forskolin alone was 24 pmol/well.
  • Results in Panels A and B correspond to the mean +/- SD of three to six independent experiments performed in quadruplicates.
  • Results in Panels C-E are representative of three similar experiments. In the figure, * means that the result was significantly different than the forskolin response, p ⁇ 0.05.
  • Figure 6 contains experiments which demonstrate that MSG inhibits cAMP accumulation in hTlRl/hTlR3-expressing cells.
  • hTlRl/hTlR3-expressing HEK293/G15 cells were induced for receptor expression as described in the methods section, (infra) Cells were incubated with 50 ⁇ M rolipram in the absence and presence of 3 mM MSG/10 mM IMP in D-PBS for 15 minutes at 37°C and cAMP levels were determined as described in the method section.
  • cAMP content of cells in the presence of rolipram was 120 pmol well. Cells were also treated with lOOng/ml PTX for 4 hours at 37°C and then stimulated as described above.
  • Panel A contains an experiment wherein hTlR2/hTlR3-expressing HEK293/G15 cells were incubated with 50 ⁇ M rolipram in the absence and presence of either ImM aspartame, 3mM cyclamate, 2mM saccharin, 50 ⁇ M monellin and 10 ⁇ M isoproterenol in D-PBS for 15 minutes at 37°C and cAMP levels were determined as described in the methods section infra. Under these conditions basal level of cAMP was 2 pmol well.
  • Panel B contains an experiment wherein hTlR2/hTlR3-expressing cells were treated with lOOng/ml PTX for 4 hours at 37°C and then stimulated as described above.
  • Panel C contains an experiment wherein mT2R5-expressing HEK293 cells were incubated with 50 ⁇ M rolipram in the absence and presence of 40 ⁇ M cycoheximide or 10 ⁇ M isoproterenol in HBSS for 15 minutes at 37°C. Under these conditions basal level of cAMP was 5 pmol/well. Cells were also treated with lOOng/ml PTX for 4 hours at 37°C and then stimulated as described above. Under these conditions basal level of cAMP was 4 pmol well. Results correspond to the mean +/- SD of three independent experiments performed in quadruplicates .
  • Figure 8 contains a schematic showing how G ⁇ i is believed to complement ⁇ -gustducin signaling pathways in TRCs. Sweet and bitter receptors functionally couple to ⁇ -gustducm (thick arrows) (10, 17) Margolskee,
  • PLC ⁇ 2 is known to be activated by the G ⁇ subunit of G proteins belonging to the Gi family (20-24) (Li et al., Science 287(54- 55):1046-9 (2000); Wu et al., Proc. Natl Acad Sci., USA 90(11):5297-5301 (1993); Katan, Biochem Biophys. Ada 1436(l-2):5-17 (1998); Smrcka et al., J. Biol Chem. 272(24):15045-48 (1993); Rhee et al., J. Biol. Chem.
  • the present invention provides cell-based assays for identifying compounds that modulate, e.g., enhance, agonize or antagonize the activity of specific TlR or T2R taste receptors or that modulate the effect of another TIR or T2R activator compound preferably by assaying their effect on the expression of an activated form of MAPK, cAMP levels or adenylyl cyclase activity by a eukaryotic cell that stably or transiently expresses at least one functional TIR or T2R.
  • the cell-based assays encompass the identification of TlR or T2R modulator by detecting its effect on any G ⁇ i associated signaling pathway.
  • the invention specifically provides cell-based assays that relate to the discovery that TlRs and T2Rs both functionally couple to G proteins other than ⁇ -gustducin or G ⁇ is, particularly Gi proteins such as G ⁇ i.
  • G proteins other than ⁇ -gustducin or G ⁇ is, particularly Gi proteins such as G ⁇ i.
  • bitter compounds such as cycloheximide specifically activate ERKl/2 mitogen activated kinases in cells expressing a T2R and G i and also that cycloheximide inhibits forskolin-induced cAMP accumulation.
  • the invention provides cell-based assays for the identification of taste modulatory compounds that rely on these discoveries. These taste modulatory compounds have potential utility as flavor enhancers or flavor additives for incorporation in foods and beverages for human or animal consumption.
  • cAMP 3' 5'-cyclic adenosine monophsphate
  • TRCs Taste receptor cells
  • GPCRs G protein-coupled receptors
  • MSG Monosodium glutamate
  • PDE phosphodiesterase
  • MAPK Mitogen activated protein kinase
  • IMP inosine monophosphate
  • PTX pertussis toxin
  • EGF Epidermal growth factor
  • PKC Protein kinase C
  • RTKs Receptor tyrosine kinases
  • PKA Protein kinase A
  • ACs Adenylyl cyclases
  • cNMP cyclic nucleotide monophosphate
  • CREB cAMP response element-binding protein
  • PLC ⁇ 2 Phospholipase C ⁇ 2
  • Trp Transient receptor potential.
  • Taste cells include neuroepithelial cells that are organized into groups to form taste buds of the tongue, e.g., foliate, fungiform, and circumvallate cells (see, e.g., Roper et al., Ann. Rev. Neurosci. 12:329-353 (1989)) (31). Taste cells are also found in the palate and other tissues, such as the esophagus and the stomach.
  • TlR refers to one or more members of a family of G protein-coupled receptors that are expressed in taste cells such as foliate, fungiform, and circumvallate cells, as well as cells of the palate, and esophagus (see, e.g., Hoon et al., Cell, 96:541-551 (1999), (32) herein incorporated by reference in its entirety).
  • TIR TIR
  • GPCR-B3 GPCR-B3
  • TRl TRl
  • GPCR-B3 is also herein referred to as rTlRl
  • GPCR-B4 is referred to as rTlR2.
  • Taste receptor cells can also be identified on the basis of morphology (see, e.g., 31), or by the expression of proteins specifically expressed in taste cells. TlR family members may have the ability to act as receptors for sweet or umami taste transduction, or to distinguish between various other taste modalities.
  • TlR sequences including hTlRl, hTlR2 and hTlR3 are identified in the Senomyx and University of California patent applications incorporated by reference in their entirety herein and are provided infra, in an Appendix after the claims.
  • TIR nucleic acids encode a family of GPCRs with seven transmembrane regions that have "G protein-coupled receptor activity,” e.g., they may bind to G proteins in response to extracellular stimuli and promote production of second messengers such as IP3, cAMP, cGMP, and Ca 2+ via stimulation of enzymes such as phospholipase C and adenylate cyclase (for a description of the structure and function of GPCRs, see, e.g., Fong, TM Cells Signal 8(3):217-224 (1996) (33) and Baldwin, et al, J. Mol. Biol 272(1):144-164 (1997) (34).
  • a single taste cell may contain many distinct TIR polypeptides.
  • TIR family therefore refers to polymorphic variants, alleles, mutants, and interspecies homologus that: (1) have at least about 35 to 50% amino acid sequence identity, optionally about 60, 75, 80, 85, 90, 95, 96, 97, 98, or 99% amino acid sequence identity to a TIR polypeptide, preferably those identified in the patent applications incorporated by reference herein, over a window of about 25 ammo acids, optionally 50-100 amino acids; (2) specifically bind to antibodies raised against an immunogen comprising an amino acid sequence preferably selected from the group consisting of the TlR polypeptide sequence disclosed in the patent applications incorporated by reference herein and conservatively modified variants thereof; (3) are encoded by a nucleic acid molecule which specifically hybridize (with a size of at least about 100, optionally at least about 500-1000 nucleotides) under stringent hybridization conditions to a sequence selected from the group consisting of the TIR nucleic acid sequences contained in the applications incorporated by reference in
  • T2R refers to one or more members of a family of G protein coupled receptors that are expressed in taste cells, specifically, the tongue and palate epithelia.
  • T2R includes the particular genes identified in the Senomyx and University of California applications relating to T2Rs incorporated by reference in their entirety herein. T2Rs are genetically linked to loci associated with bitter taste perception in mice and humans.
  • T2R and terms including T2R, e.g., T2R04 or T2R05 refers generally to isolated T2R nucleic acids, isolated polypeptides encoded by T2R nucleic acids, and activities thereof. T2R nucleic acids and polypeptides can be derived from any organism.
  • T2R and terms including “T2R” also refer to polypeptides comprising receptors that are activated by bitter compounds, and to nucleic acids encoding the same. Thus both TlRs and T2Rs comprise different families of chemosensory GPCRs. Sequences of various T2Rs are also contained in the Appendix that precedes the claims.
  • G proteins are heterotrimeric proteins composed of a single ⁇ subunit complexed with the ⁇ dimer. Molecular cloning has resulted in the identification of 18 distinct . ⁇ . subunits, 5 ⁇ subunits, and 12 ⁇ subunits. G proteins are usually divided into four subfamilies Gi, G s , G q , and G12 based on the sequence similarity of the G ⁇ subunit.
  • Gi, G s , G q , and G12 based on the sequence similarity of the G ⁇ subunit.
  • chemosensory GPCRs have an "N-terminal domain;” “extracellular domains;” “transmembrane domains” comprising seven transmembrane regions, and corresponding cytoplasmic, and extracellular loops; “cytoplasmic domains,” and a "C-terminal domain” (see, e.g., Hoon et al, Cell, 96:541-551 (1999) (115); Buck & Axel, Cell, 65:175-187 (1991)) (44).
  • domains can be structurally identified using methods known to those of skill in the art, such as sequence analysis programs that identify hydrophobic and hydrophilic domains (see, e.g., Stryer, Biochemistry, (3rd ed. 1988) (45); see also any of a number of Internet based sequence analysis programs. Such domains are useful for making chimeric proteins and for in vitro assays of the invention, e.g., ligand binding assays.
  • Extracellular domains therefore refers to the domains of TIR and
  • T2R polypeptides that protrude from the cellular membrane and are exposed to the extracellular face of the cell.
  • Such domains generally include the "N terminal domain” that is exposed to the extracellular face of the cell, and optionally can include portions of the extracellular loops of the transmembrane domain that are exposed to the extracellular face of the cell, i.e., the loops between transmembrane regions 2 and 3, between transmembrane regions 4 and
  • the "N-terminal domain” region starts at the N-terminus and extends to a region close to the start of the first transmembrane domain. More particularly, in one embodiment of the invention, this domain starts at the N- terminus and ends approximately at the conserved glutamic acid at amino acid position 563 plus or minus approximately 20 amino acids.
  • These extracellular domains are useful for in vitro ligand-binding assays, both soluble and solid phase.
  • transmembrane regions, described below can also bind ligand either in combination with the extracellular domain, and are therefore also useful for in vitro ligand-binding assays.
  • Transmembrane domain which comprises the seven “transmembrane regions,” refers to the domain of TlR or T2R polypeptides that lies within the plasma membrane, and may also include the corresponding cytoplasmic (intracellular) and extracellular loops. In one embodiment, this region corresponds to the domain of TIR or T2R family members. In the case of TlR family member this starts approximately at the conserved glutamic acid residue at amino acid position 563 plus or minus 20 amino acids and ends approximately at the conserved tyrosine amino acid residue at position 812 plus or minus approximately 10 amino acids.
  • the seven transmembrane regions and extracellular and cytoplasmic loops can be identified using standard methods, as described in Kyte & Doolittle, J. Mol Biol, 157:105-32 (1982)) (46), or in Stryer, supra (45).
  • Cytoplasmic domains refers to the domains of TlR or T2R polypeptides that face the inside of the cell, e.g., the "C-terminal domain” and the intracellular loops of the transmembrane domain, e.g., the intracellular loop between transmembrane regions 1 and 2, the intracellular loop between transmembrane regions 3 and 4, and the intracellular loop between transmembrane regions 5 and 6.
  • C-terminal domain refers to the region that spans the end of the last transmembrane domain and the 0-terminus of the protein, and which is normally located within the cytoplasm. In one embodiment, this region starts at the conserved tyrosine amino acid residue at position 812 plus or minus approximately 10 amino acids and continues to the C- ter minus of the polypeptide.
  • ligand-binding region refers to sequences derived from a taste receptor, particularly a taste receptor that substantially incorporates at least the extracellular domain of the receptor.
  • the extracellular domain of the ligand-binding region may include the N-terminal domain and, optionally, portions of the transmembrane domain, such as the extracellular loops of the transmembrane domain.
  • the ligand-binding region may be capable of binding a ligand, and more particularly, a compound that enhances, mimics, blocks, and/or modulates taste, e.g., sweet, bitter, or umami taste.
  • T2Rs the compound bound by the ligand binding region will modulate bitter taste.
  • TlRs the compound bound by the ligand-binding region will modulate sweet or umami taste.
  • heteromultimer or “heteromultimeric complex” in the context of the TIR receptors or polypeptides used in the assays of the present invention refers to a functional association of at least one TIR receptor and another receptor, typically another TlR receptor polypeptide (or, alternatively another non-TlR receptor polypeptide).
  • another receptor typically another TlR receptor polypeptide (or, alternatively another non-TlR receptor polypeptide).
  • TlR receptor polypeptide or, alternatively another non-TlR receptor polypeptide
  • T1R3 may function solely to facilitate surface expression of TlRl and T1R2, which may act independently as taste receptors.
  • a functional taste receptor may be comprised solely of T1R3, which is differentially processed under the control of TlRl or T1R2, analogous to RAMP-dependent processing of the calcium-related receptor.
  • T2Rs the eukaryotic cells used in the subject MAPK assays will preferably express a single T2R.
  • modulator or “modulatory compound” means any compound that itself affects the activity of a TlR or T2R or modulates (affects) the effect of another compound on TIR or T2R activity.
  • modulation is determined by cell-based assays that detect the effect of a putative modulator or Gi signaling pathways, e.g., assays that detect the effect of a compound on MAPK activity, cAMP levels or adenylyl cyclase activity.
  • the phrase "functional effects" in the context of for testing compounds that modulate at least one TlR or T2R family member mediated taste transduction includes the determination of any parameter that is indirectly or directly under the influence of the receptor, e.g., functional, physical and chemical effects. It includes ligand binding, changes in ion flux, membrane potential, current flow, transcription, G protein binding, GPCR phosphorylation or dephosphorylation, conformation change-based assays, signal transduction, receptor-ligand interactions, second messenger concentrations (e.g., cAMP, cGMP, IP3, or intracellular Ca 2+ ), in vitro, in vivo, and ex vivo and also includes other physiologic effects such increases or decreases of neurotransmitter or hormone release.
  • functional, physical and chemical effects includes the determination of any parameter that is indirectly or directly under the influence of the receptor, e.g., functional, physical and chemical effects. It includes ligand binding, changes in ion flux, membrane potential, current flow, transcription, G protein binding, GPCR
  • the assays will generally measure the effect of a compound on MAPK activation, cAMP accumulation or adenylyl cyclase activity in cell-based expression systems whereby the TIR or T2R is functionally coupled to a Gi protein such as G ⁇ i and the assays are used to screen for putative sweeteners or sweet taste modulators or enhancers, umami taste modulators or enhancers, or bitter compounds or bitter taste modulators or enhancers, e.g., bitter taste blockers.
  • Such modulators have application for incorporation in foods, beverages, pharmaceuticals, and the like for human or animal consumption.
  • determining the functional effect in the context of assays is meant assays for a compound that increases or decreases a parameter that is indirectly or directly under the influence of at least one TIR or T2R family member, e.g., functional, physical and chemical effects.
  • Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbency, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties, patch clamping, voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers, oocyte TlR or T2R gene expression; tissue culture cell TlR or T2R expression; transcriptional activation of TIR or T2R genes; ligand-binding assays; voltage, membrane potential and conductance changes; ion flux assays; changes in intracellular second messengers such as cAMP, cGMP, and inositol triphosphate (IP3); changes in intracellular calcium levels; neurotransmitter release, conformational assays and the like.
  • the effect of a putative modulator compound will be preferably assayed based on its effect on MAPK activation, cAMP accumulation,
  • Inhibitors "inhibitors,” “activators,” “enhancer,” and “modulators” of TlR or T2R genes or proteins are used to refer to inhibitory, activating, or modulating molecules identified using in vitro and in vivo assays for taste transduction, e.g., ligands, agonists, antagonists, inversed agonists, and their homologues and mimetics. These compounds themselves modulate TlR or T2R activity or modulate the effect of another compound on TlR or T2R activity. In the present invention these molecules will preferably be identified using the subject cell- based MAPK or cAMP assays. In preferred embodiments, the "inhibitors” will block taste of a known bitter compound or enhance the taste of a known sweet or umami compound or compounds.
  • Inhibitors are compounds that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate taste transduction, e.g., antagonists.
  • Activators are compounds that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize, or up regulate taste transduction, e.g., agonists.
  • Modulators include compounds that, e.g., alter the interaction of a receptor with: extracellular proteins that bind activators or inhibitor (e.g., ebnerin and other members of the hydrophobic carrier family); G proteins; kinases (e.g., homologues of rhodopsin kinase and beta adrenergic receptor kinases that are involved in deactivation and desensitization of a receptor); and arrestins, which also deactivate and desensitize receptors.
  • Modulators can include genetically modified versions of TIR or T2R family members, e.g., with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • Such assays for inhibitors and activators include, e.g., expressing TIR or T2R family members in cells or cell membranes, applying putative modulator compounds, in the presence or absence of tastants, e.g., sweet, umami or bitter tastants, and then determining the functional effects on taste transduction, as described above.
  • Samples or assays comprising TIR or T2R family members that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of modulation.
  • Positive control samples e.g. a sweet, umami, or bitter tastant without added modulators
  • Negative control samples e.g., buffer without an added taste stimulus
  • a relative TIR or T2R activity value 0%. Inhibition of a TlR or
  • T2R is achieved when a mixture of the positive control sample and a modulator result in the TIR or T2R activity value relative to the positive control is about
  • Activation of a TIR or T2R by a modulator alone is achieved when the TlR activity value relative to the positive control sample is 10%, 25%, 50%, 75%, optionally 100%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
  • purified refers to the state of being free of other, dissimilar compounds with which the compound of the invention is normally associated in its natural state, so that the “purified,” “substantially purified,” and “isolated” subject comprises at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably at least 50% or 75% of the mass, by weight, of a given sample. In one preferred embodiment, these terms refer to the compound of the invention comprising at least 95% of the mass, by weight, of a given sample.
  • the terms “purified,” “substantially purified,” and “isolated,” when referring to a nucleic acid or protein, also refers to a state of purification or concentration different than that which occurs naturally in the mammalian, especially human body.
  • nucleic acid or protein or classes of nucleic acids or proteins, described herein may be isolated, or otherwise associated with structures or compounds to which they are not normally associated in nature, according to a variety of methods and processes known to those of skill in the art.
  • nucleic acid or “nucleic acid sequence” refers to a deoxy- ribonucleotide or ribonucleotide oligonucleotide in either single- or double- stranded form.
  • the term encompasses nucleic acids, i.e., oligonucleotides, containing known analogs of natural nucleotides.
  • the term also encompasses nucleic-acid- like structures with synthetic backbones (see e.g., Oligonucleotides and Analogues, a Practical Approach, ed. F. Eckstein, Oxford Univ. Press (1991); Antisense Strategies, Annals of the N. Y. Academy of Sciences, Vol.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating, e.g., sequences in which the third position of one or more selected codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al, J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al, Mol. Cell. Probes, 8:91-98 (1994)) (54-56).
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • Plasmid membrane translocation domain or simply “translocation domain” means a polypeptide domain that, when incorporated into a polypeptide coding sequence, can with greater efficiency “chaperone” or “translocate” the hybrid ("fusion") protein to the cell plasma membrane than without the domain.
  • a “translocation domain” may be derived from the amino terminus of the bovine rhodopsin receptor polypeptide, a 7- transmembrane receptor.
  • rhodopsin from any mammal may be used, as can other translocation facilitating sequences.
  • the translocation domain is particularly efficient in translocating 7-transmembrane fusion proteins to the plasma membrane, and a protein (e.g., a taste receptor polypeptide) comprising an amino terminal translocating domain will be transported to the plasma membrane more efficiently than without the domain.
  • a protein e.g., a taste receptor polypeptide
  • the use of other translocation domains may be preferred.
  • a PDZ domain-interacting peptide as described herein, may be used.
  • the "translocation domain,” “ligand-binding domain”, and chimeric receptors compositions described herein also include “analogs,” or “conservative variants” and “mirnetics” ("peptidomimetics") with structures and activity that substantially correspond to the exemplary sequences.
  • the terms “conservative variant” or “analog” or “mimetic” refer to a polypeptide which has a modified amino acid sequence, such that the change(s) do not substantially alter the polypeptide's (the conservative variant's) structure and/or activity, as defined herein.
  • amino acid sequence i.e., amino acid substitutions, additions or deletions of those residues that are not critical for protein activity, or substitution of amino acids with residues having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids does not substantially alter structure and/or activity.
  • conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences . Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.
  • the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein, which encodes a polypeptide, also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid, which encodes a polypeptide, is implicit in each described sequence.
  • one exemplary guideline to select conservative substitutions includes (original residue followed by exemplary substitution): ala gly or ser; arg/lys; asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gin; ile/leu or val; leu/ile or val; lys/arg or gin or glu; met/leu or tyr or lie; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe; val/ile or leu.
  • An alternative exemplary guideline uses the following six groups, each containing amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (I); 5) Isoleueine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (see also, e.g., Creighton, Proteins, W.H.
  • mimetic and “peptidomimetic” refer to a synthetic chemical compound that has substantially the same structural and/or functional characteristics of the polypeptides, e.g., translocation domains, ligand-binding domains, or chimeric receptors of the invention.
  • the mimetic can be either entirely composed of synthetic, non-natural analogs of amino acids, or may be a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
  • the mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity.
  • Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet,
  • a polypeptide can be characterized as a
  • glutar aldehyde N-hydroxysuccinimide esters, bifunctional maleimides, N,N'dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC).
  • DCC N,N'dicyclohexylcarbodiimide
  • DIC N,N'-diisopropylcarbodiimide
  • linkages include, e.g., ketomethylene ie.g.,
  • a polypeptide can also be any polypeptide
  • a “label” or a “detectable moiety” is a composition detectable by
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents,
  • enzymes e.g., as .commonly used in an ELISA
  • biotin e.g., as .commonly used in an ELISA
  • digoxigenin e.g., digoxigenin
  • haptens e.g., haptens
  • proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
  • a "labeled nucleic acid probe or oligonucleotide” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
  • nucleic acid probe or oligonucleotide is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • a probe may include natural (i.e., A, G, C, or T) or modified bases (7- deazaguanosine, inosine, etc.).
  • the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
  • probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.
  • probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
  • the probes are optionally directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
  • heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
  • a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
  • a “promoter” is defined as an array of nucleic acid sequences that direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • a “constitutive” promoter is a promoter that is active under most environmental and developmental conditions.
  • an "inducible" promoter is a promoter that is active under environmental or developmental regulation.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • recombinant refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide ("recombinant protein") encoded by a recombinant polynucleotide.
  • Recombinant means also encompass the ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for expression of, e.g., inducible or constitutive expression of a fusion protein comprising a translocation domain of the invention and a nucleic acid sequence amplified using a primer of the invention.
  • a “stable cell line” refers to a cell line, which stably, i.e. over a prolonged period, expresses a heterologous nucleic sequence, i.e., a TIR, T2R or G protein.
  • a heterologous nucleic sequence i.e., a TIR, T2R or G protein.
  • such stable cell lines will be produced by transfecting appropriate cells, typically mammalian cells, e.g. HEK- 293 cells, with a linearized vector that contains a TIR or T2R expression construct that expresses at least one TlR or T2R, i.e., TlRl, T1R2 and/or T1R3 or a T2R.
  • such stable cell lines that express a functional TIR or T2R receptor will be produced by co-transfecting two linearized plasmids that express hTlRl and hTIRS or hTlR2 and hTlR3 or a single line plasmid that expresses a specific T2R and an appropriate selection procedure to generate cell lines having these genes stably integrated therein.
  • the cell line will also stably express a G protein preferably a Gi such as G ⁇ i or G ⁇ i5.
  • Gi such as G ⁇ i or G ⁇ i5.
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms "variable light chain” (VL) and “variable heavy chain” (VH) refer to these light and heavy chains respectively.
  • a "chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • An “anti-TlR” antibody is an antibody or antibody fragment that specifically binds a polypeptide encoded by a TIR gene, cDNA, or a subsequence or variant thereof.
  • an "anti-T2R” antibody is an antibody or antibody fragment that specifically binds a polypeptide encoded by T2R gene, cDNA, or a subsequence or variant thereof.
  • an "anti-activated MAPK antibody” or an “anti-phospho MAPK antibody” refers to an antibody or antibody fragment that specifically binds to an activated (phosphorylated) form of MAPK.
  • a "ligand that detects cAMP” is any moiety that specifically detects cAMP levels.
  • immunoassay is an assay that uses an antibody to specifically bind an antigen.
  • the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
  • MAPK activity or cAMP levels will be immunoassayed in eukaryotic cells using an antibody that specifically recognizes an activated form of MAPK or cAMP.
  • the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • polyclonal antibodies raised to a TIR or T2R family member from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the TIR or T2R polypeptide or an immunogenic portion thereof and not with other proteins, except for orthologs or polymorphic variants and alleles of the TIR or T2R polypeptide.
  • This selection may be achieved by subtracting out antibodies that cross-react with TIR or T2R molecules from other species or other TIR or T2R molecules.
  • Antibodies can also be selected that recognize only TIR GPCR family members but not GPCRs from other families. In the case of antibodies to activated MAPKs, suitable polyclonal and monoclonal antibodies are commercially available.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Hariow & Lane, Antibodies, A Laboratory Manual, (1988) (59), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • expression vector refers to any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell.
  • the term includes linear or circular expression systems.
  • the term includes expression systems that remain episomal or integrate into the host cell genome.
  • the expression systems can have the ability to self-replicate or not, i.e., drive only transient expression in a cell.
  • the term includes recombinant expression "cassettes which contain only the minimum elements needed for transcription of the recombinant nucleic acid.
  • host cell is meant a cell that contains an expression vector and supports the replication or expression of the expression vector.
  • Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, worm or mammalian cells such as CHO, Hela, BHK, HEK-293, and the like, e.g., cultured cells, explants, and cells in vivo.
  • nucleotide or protein length an amount of binding, etc. is meant to encompass variations of ⁇ 20% or +10%, more preferably ⁇ 5%, even more preferably ⁇ 1, and still more preferably +.1% from the specified amount, as such variations are appropriate to perform a disclosed method or otherwise carry out the present invention.
  • nucleotide sequences refers to two or more sequences that have at least about least 60%, preferably at least about 70%, more preferably at least about 80%, more preferably about 90% to 99%, still more preferably about 95% to about 99%, and most preferably about 99% nucleotide identify, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the substantial identity exists in nucleotide sequences of at least about 100 residues, more preferably in nucleotide sequences of at least about 150 residues, and most preferably in nucleotide sequences comprising a full length coding sequence.
  • full length is used herein to refer to a complete open reading frame encoding a functional TIR or T2R polypeptide, as described further herein below. Methods for determining percent identity between two polypeptides are defined herein below under the heading "Nucleotide and Amino Acid Sequence Comparisons”.
  • substantially identical sequences can be polymorphic sequences.
  • the term "polymorphic" refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. An allelic difference can be as small as one base pair.
  • substantially identical sequences can comprise mutagenized sequences, including sequences comprising silent mutations. A mutation can comprise one or more residue changes, a deletion of residues, or an insertion of additional residues.
  • nucleic acid sequences are substantially identical.
  • two nucleic acid sequences being compared can be designated a "probe” and a "target.”
  • a “probe” is a reference nucleic acid molecule
  • a "target” is a test nucleic acid molecule, often found within a heterogeneous population of nucleic acid molecules.
  • a “target sequence” is synonymous with a "test sequence.”
  • a preferred nucleotide sequence employed for hybridization studies or assays includes probe sequences that are complementary to or mimic at least an about 14 to 40 nucleotide sequence of a nucleic acid molecule of the present invention.
  • probes comprise 14 to 20 nucleotides, or even longer where desired, such as 30, 40, 50, 60, 100, 200, 300, or 500 nucleotides or up to the full length of the particular TlR or T2R.
  • Such fragments can be reddily prepared by, for example, chemical synthesis of the fragment, by application of nucleic acid amplification technology, or by introducing selected sequences into recombinant vectors for recombinant production.
  • hybridizing specifically to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex nucleic acid mixture (e.g., total cellular DNA or RNA).
  • the phrase "selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
  • stringent hybridization conditions and “stringent hybridization wash conditions” refer to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is that in Tigssen, Techniques in Biochemistry and Molecular
  • Tm thermal melting point
  • Stringent conditions will be those in which the salt concentration is less than about 1.0M sodium ion, typically about 0.01 to 1.0M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the additional of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, optionally 10 times background hybridization. Exemplary stringent hybridization conditions are:
  • hybridization and wash steps effected in said exemplary stringent hybridization conditions are each effected for at least 1, 2, 5, 10, 15, 30, 60, or more minutes.
  • the wash and hybridization steps are each effected for at least 5 minutes, and more preferably, 10 minutes, 15 minutes, or more than 15 minutes.
  • hybridizing substantially to refers to complementary hybridization between a probe nucleic acid molecule and a target nucleic acid molecule and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired hybridization.
  • An example of stringent hybridization conditions for Southern or Northern Blot analysis of complementary nucleic acids having more than about 100 complementary residues is overnight hybridization in 50% formamide with 1 mg of heparin at 42°C.
  • An example of highly stringent wash conditions is 15 minutes in 0.1X SSC at 65°C.
  • An example of stringent wash conditions is 15 minutes in 0.2X SSC buffer at 65°C. See Sambrook et al., eds (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (60) for a description of SSC buffer. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example of medium stringency wash conditions for a duplex of more than about 100 nucleotides is 15 minutes in IX SSC at 45°C.
  • An example of low stringency wash for a duplex of more than about 100 nucleotides is 15 minutes in 4X to 6X SSC at 40°C.
  • stringent conditions typically involve salt concentrations of less than about 1 M Na + ion, typically about 0.01 to 1 M Na + ion concentration (or other salts) at pH 7.0-8.3, and the temperature is typically at least about 30°C.
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2-fold (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • a probe nucleotide sequence preferably hybridizes to a target nucleotide sequence in 7% sodium dodecyl sulphate (SDS), 0.5M NaP0 , 1 mM EDTA at 50°C followed by washing in 2X SSC, 0.1% SDS at 50°C; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulphate (SDS), 0.5M NaP0 , 1 mM EDTA at 50°C followed by washing in IX SSC, 0.1% SDS at 50°C; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulphate (SDS), 0.5M NaP04, 1 MM EDTA at 50°C followed by washing in 0.5X SSC, 0.1% SDS at 50°C;
  • nucleic acid sequences are substantially identical, share an overall three-dimensional structure, or are biologically functional equivalents. Nucleic acid molecules that do not hybridize to each other under stringent conditions are still substantially identical if the corresponding proteins are substantially identical. This can occur, for example, when two nucleotide sequences comprise conservatively substituted variants as permitted by the genetic code.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially related if the polypeptides that they encode are substantially related. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary "moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCI, 1% SDS at 37°C, and a wash in IX SSC at 45°C. Such hybridizations and wash steps can be carried out for, e.g., 1, 2, 5, 10, 15, 30, 60, or more minutes. Preferably, the wash and hybridization steps are each effected for at least 5 minutes. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
  • TIR or T2R also encompasses nucleic acids comprising subsequences and elongated sequences of a TlR or T2R nucleic acid, including nucleic acids complementary to a TIR or T2R nucleic acid, TlR or T2R RNA molecules, and nucleic acids complementary to TlR or T2R RNAs (cRNAs).
  • cRNAs TlR or T2R RNAs
  • sequence refers to a sequence of nucleic acids that comprises a part of a longer nucleic acid sequence.
  • An exemplary subsequence is a probe, described herein above, or a primer.
  • primer refers to a contiguous sequence comprising about 8 or more deoxyribonucleotides or ribonucleotides, preferably 10-20 nucleotides, and more preferably 20-30 nucleotides of a selected nucleic acid molecule.
  • the primers of the invention encompass oligonucleotides of sufficient length and appropriate sequence so as to provide initiation of polymerization on a nucleic acid molecule of the present invention.
  • the term "elongated sequence” refers to an addition of nucleotides (or other analogous molecules) incorporated into the nucleic acid.
  • a polymerase e.g., a DNA polymerase
  • the nucleotide sequence can be combined with other DNA sequences, such as promoters, promoter regions, enhancers, polyadenylation signals, intronic sequences, additional restriction enzyme sites, multiple cloning sites, and other coding segments.
  • complementary sequences indicates two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between base pairs.
  • complementary sequences means nucleotide sequences which are substantially complementary, as can be assessed by the same nucleotide comparison methods set forth below, or is defined as being capable of hybridizing to the nucleic acid segment in question under relatively stringent conditions such as those described herein.
  • a particular example of a complementary nucleic acid segment is an antisense oligonucleotide.
  • gene refers broadly to any segment of DNA associated with a biological function.
  • a gene encompasses sequences including but not limited to a coding sequence, a promoter region, a cis-regulatory sequence, a non-expressed DNA segment that is a specific recognition sequence for regulatory proteins, a non-expressed DNA segment that contributes to gene expression, a DNA segment designed to have desired parameters, or combinations thereof.
  • a gene can be obtained by a variety of methods, including cloning from a biological sample, synthesis based on known or predicted sequence information, and recombinant derivation of an existing sequence.
  • chimeric gene refers to a promoter region operatively linked to a TlR or T2R sequence, including a TIR or T2R cDNA, a TIR or T2R nucleic acid encoding an antisense RNA molecule, a TlR or T2R nucleic acid encoding an RNA molecule having tertiary structure (e.g., a hairpin structure) or a TIR or T2R nucleic acid encoding a double-stranded RNA molecule.
  • chimeric gene also refers to a TIR or T2R promoter region operatively linked to a heterologous sequence.
  • operatively linked refers to a functional combination between a promoter region and a nucleotide sequence such that the transcription of the nucleotide sequence is controlled and regulated by the promoter region. Techniques for operatively linking a promoter region to a nucleotide sequence are known in the art.
  • vector is used herein to refer to a nucleic acid molecule having nucleotide sequences that enable its replication in a host cell.
  • a vector can also include nucleotide sequences to permit ligation of nucleotide sequences within the vector, wherein such nucleotide sequences are also replicated in a host cell. Representative vectors include plasmids, cosmids, and viral vectors.
  • a vector can also mediate recombinant production of a TlR or T2R polypeptide, as described further herein below.
  • construct refers to a vector further comprising a nucleotide sequence operatively inserted with the vector, such that the nucleotide sequence is recombinantly expressed.
  • recombinantly expressed or “recombinantly produced” are used interchangeably to refer generally to the process by which a polypeptide encoded by a recombinant nucleic acid is produced.
  • heterologous nucleic acids refers to a sequence that originates from a source foreign to an intended host cell or, if from the same source, is modified from its original form.
  • recombinant TIR or T2R nucleic acids comprise heterologous nucleic acids.
  • a heterologous nucleic acid in a host cell can comprise a nucleic acid that is endogenous to the particular host cell but has been modified, for example by mutagenesis or by isolation from native cis -regulatory sequences.
  • a heterologous nucleic acid also includes non-naturally occurring multiple copies of a native nucleotide sequence.
  • a heterologous nucleic acid can also comprise a nucleic acid that is incorporated into a host cell's nucleic acids at a position wherein such nucleic acids are not ordinarily found.
  • Nucleic acids used in the cell-based assays of the present invention preferably MAPK and cAMP assays can be cloned, synthesized, altered, mutagenized, or combinations thereof.
  • Standard recombinant DNA and molecular cloning techniques used to isolate nucleic acids are known in the art.
  • Site-specific mutagenesis to create base pair changes, deletions, or small insertions are also known in the art. See e.g., Sambrook et al. (eds.) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); Silhavy et al. Experiments with Gene Fusions.
  • substantially identical refers to a sequence that is at least about 35% identical to the particular TIR or T2R protein, when compared over the full length of the TIR or T2R protein.
  • a protein substantially identical to the TIR or T2R protein used in the present invention comprises an amino acid sequence that is at least about 35% to about 45% identical to a particular TIR or
  • T2R more preferably at least about 45% to about 55% identical thereto, even more preferably at least about 55% to about 65% identical thereto, still more preferably at least about 65% to about 75% identical thereto, still more preferably at least about 75% to about 85% identical thereto, still more preferably at least about 85% to about 95% identical thereto, and still more preferably at least about 95% to about 99% identical thereto when compared over the full length of the particular TIR or T2R.
  • the term "full length” refers to a functional TIR or T2R polypeptide. Methods for determining percent identity between two polypeptides are also defined herein below under the heading "Nucleotide and Amino Acid Sequence Comparisons".
  • substantially identical when used to describe polypeptides, also encompasses two or more polypeptides sharing a conserved three- dimensional structure.
  • Computational methods can be used to compare structural representations, and structural models can be generated and easily tuned to identify similarities around important active sites or ligand binding sites. See Saqi et al. Bioinformatics 15:521-522 (1999); Barton Acta Crystallogr D Biol Crystallogr 54:1139-1146 (1998); Henikoff et al. Electrophoresis 21:1700- 1706 (2000); and Huang et al. Pac Symp Biocomput:2S0-24:l (2000) (64-67).
  • Substantially identical proteins also include proteins comprising amino acids that are functionally equivalent to a TIR or T2R according to the invention.
  • the term "functionally equivalent” in the context of amino acids is known in the art and is based on the relative similarity of the amino acid side-chain substituents. See Henikoff & Henikoff Adv Protein Chem 54:73-97 (2000) (68).
  • arginine, lysine, and histidine are all positively charged residues; that alanine, glycine, and serine are all of similar size; and that phenylalanine, tryptophan, and tyrosine all have a generally similar shape.
  • arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine are defined herein as biologically functional equivalents.
  • hydropathic index of amino acids can be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • substantially identical also encompasses polypeptides that are biologically functional equivalents of a particular TIR or T2R polypeptide.
  • the term "functional" includes an activity of an TIR or T2R polypeptide, for example activating intracellular signaling pathways (e.g., coupling with gustducin) and mediating taste perception. Preferably, such activation shows a magnitude and kinetics that are substantially similar to that of a cognate TlR or T2R polypeptide in vivo. Representative methods for assessing TlR or T2R activity are described in the patent applications incorporated by reference herein.
  • the assays of the present invention also can use functional fragments of a particular TlR or T2R polypeptide. Such functional portion need not comprise all or substantially all of the amino acid sequence of a native TlR or T2R gene product.
  • the assays of the present invention also can use functional polypeptide sequences that are longer sequences than that of a native TlR or T2R polypeptide. For example, one or more amino acids can be added to the N- terminus or C-terminus of a TlR or T2R polypeptide. Such additional amino acids can be employed in a variety of applications, including but not limited to purification applications. Methods of preparing elongated proteins are known in the art.
  • MAPK or 'MAP Kinase refers to a mitogen activated protein kinase, the expression of which is activated by some functional GPCRs, i.e., T2Rs and TlRs.
  • MAPK' or 'MAP Kinase activation specific ligands refers to a ligand, preferably a polyclonal or monoclonal antibody or fragment thereof that specifically binds an activated form of MAPK, e.g., p42/p44 MAPK or p38/MAPK.
  • Antibodies that specifically bind the activated (phosphorylated) form of MAPK are commercially available and include the phosph-p44/p42 MAP Kinase antibody #9106 available from Cell Signaling Technologies, the polyclonal anti- phospho-p44/42 MAPK and anti-phospho-p38 MAPK antibodies available from UBI, (Lake Placid, NY, USA) and New England Biolabs (Beverly, MA, USA), the anti-phospho-p44/42 MAPK antibodies reported by Discovery Research Laboratories III, Takeda Chemical Indust. Ltd., (Oskaka Japan) (Tan et al., J. Immunol. Meth. 232(1-2): 87-97 (1998)) (70).
  • Ligand or “compound” that "activates MAPK” refers to a compound which when contacted with a eukaryotic cell that expresses a functional GPCR, herein at least one functional TlR or T2R, results in a detectable increase in the activated form of MAPK. This increase will preferably will be detected by antibody-based detection methods that use an antibody that specifically binds to an activated form of MAPK.
  • PLC refers to phospholipase C.
  • a ligand or compound that activates MAPK may activate MAPK in cells via a pathway that is independent of PLC activation.
  • present invention generally relates to cell-based assays for identifying compounds that modulate the activity of at least one TIR or T2R taste receptor, wherein the assays comprise contacting a eukaryotic cell that stably or transiently expresses at least one functional TIR or T2R and a G protein that functionally couples therewith, e.g.
  • Gi protein such as G ⁇ i with a putative modulator of said functional TIR or T2R, and assaying the effect of said putative agonist or antagonist compound on G mediated signaling pathways, e.g., by assaying the effect of said putative modulation on MAPK activation, cAMP accumulation or adenylyl cyclase activity.
  • a modulator compound will result, e.g., in a detectable increase or decrease in the amount of an activated form of MAPK, i.e., phosphorylated MAPK, e.g., phosphorylated p44/42 MAP Kinase or phosphorylated p38 MAP Kinase, and will elicit this effect on MAPK activation by a pathway independent of PLC activation or will result in detectable increase or decrease in cAMP accumulation, or will result in a change (e.g., decrease) in adenylyl cyclase activity.
  • the invention embraces any cell-based assays that identify compounds that modulate to a TRGPCR (TlR or T2R)/G ⁇ i mediated signaling pathway.
  • the eukaryotic cells used in the subject assays will stably or transiently express at least one functional TIR or T2R.
  • the eukaryotic cell will either stably or transiently express a functional T1R1/T1R3 umami taste receptor or a functional T1R2/T1R3 sweet taste receptor or will stably or transiently express a desired functional T2R, preferably a functional human TIR or T2R taste receptor.
  • the eukaryotic cell will further be transfected to stably or transiently express or will endogenously express a G protein that couples with said TlR(s) or T2R thereby resulting in a functional taste receptor.
  • a G protein that couples with said TlR(s) or T2R thereby resulting in a functional taste receptor.
  • suitable G proteins are known in the art and are referred in the patent applications incorporated by reference herein.
  • the G protein will comprise a Gi protein selected from G ⁇ i, i.e.
  • the G protein will comprise G ⁇ is, ⁇ -transcucin, gustducin, G ⁇ z or a functional chimera or variant thereof that couples with the TlR(s) or T2R expressed by the eukaryotic cell.
  • the present assays can be effected, using any eukaryotic cell that functionally expresses the particular TlR(s) or T2R, and which cell, when contacted with an activator of said TIR or TIR results in an increase in an activated form of MAPK, or a decrease in cAMP accumulation or a reduction in adenylyl cyclase activity by a pathway that is independent of PLC activation.
  • suitable eukaryotic cells include amphibian, yeast, insect, amphibian, worm and mammalian cells.
  • suitable cells for use in the subject cell-based assays include HEK293 cells, BHK cells, CHO cells, Hela cells and Xenopus oocytes,.
  • the eukaryotic cells used in the subject cell- based assays will comprise HEK293 cells that stably or transiently express at least one or functional TlR or T2R taste receptor by the transfection of such cells with a cDNA or cDNAs encoding said at least one TIR or T2R.
  • HEK293 cells stably expressing the large T cell antigen and the promiscuous G protein G ⁇ is (HEK293T-G ⁇ i5) or G ⁇ i can be transiently transfected with a particular taste receptor plasmid by known transfection methods, e.g., by use of Ca 2+ phosphate or lipid-based systems, or other transformation methods referenced supra.
  • the TlR or T2R expressing cell will further express endogenously or be engineered to express a G protein that functionally couples therewith, e.g., a G protein selected from the G ⁇ i proteins identified previously.
  • Cells that stably or transiently express the particular taste receptor are used in assays that measure the effect of at least one putative TIR or T2R modulatory compound on G ⁇ i ⁇ mediated signaling pathways, e.g., by measuring its effect on MAPK activation, cAMP accumulation or adenylyl cyclase activity.
  • the MAPK or cAMP assays of the present invention can use immobilized cells or cells in suspension.
  • the taste receptor expressing cells will be seeded into multi-well culture plates, e.g., 6-well culture plates.
  • other in vitro cell culture devices can be substituted therefore, and is not critical to the invention.
  • TlR or T2R expressing eukaryotic cell functional expression of the TlR or T2R expressing eukaryotic cell is allowed to proceed for a certain time, e.g., on the order of about 48 hours, and then taste receptor expressing cells are stimulated with a putative modulatory compound for a fixed time, e.g., about 5 minutes, and then the reaction is then stopped, e.g., by the addition of ice-cold buffer, and the cells are then assayed for changes in activated MAPK, cAMP or adenylyl cyclase activity.
  • these reaction times may be shortened or lengthened within wide limits.
  • the level of activated MAPK produced by such cells is detected in whole cells or cell lysates.
  • cell lysates are prepared by known methods, and detected by activated cAMP, MAPK or adenylyl cyclase activity is detected by known methods.
  • activated MAPK can be the use of a polyclonal or monoclonal antibody or fragment thereof that specifically recognizes an activated (phosphorylated) form of MAPK.
  • activation of MAPK is detected by Western analysis of cell lysates using a specific monoclonal antibody that recognizes phosphorylated (active) MAPK (Phospho-p44/42 MAP Kinase antibody #9106 available from Cell Signaling Technologies) or another commercially available antibody that specifically recognizes activated MAPK.
  • a specific monoclonal antibody that recognizes phosphorylated (active) MAPK Phospho-p44/42 MAP Kinase antibody #9106 available from Cell Signaling Technologies
  • another commercially available antibody that specifically recognizes activated MAPK.
  • a measure of receptor activity is the binding of GTP by cell membranes containing receptors.
  • membranes isolated from cells expressing the receptor are incubated in a buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCI, and 10 mM MgCl 2 , 80 pM . ⁇ S-GTP ⁇ S and 3 ⁇ M GDP.
  • the assay mixture is incubated for 60 minutes at 30°C, after which unbound labelled GTP is removed by filtration onto GF/B filters. Bound, labelled GTP is measured by liquid scintillation counting. The presence and absence of a candidate modulator of TIR or T2R activity. A decrease of 10% or more in labelled GTP binding as measured by scintillation counting in an assay of this kind containing a candidate modulator, relative to an assay without the modulator, indicates that the candidate modulator inhibits TIR or T2R activity.
  • a compound is considered an agonist if it induces at least 50% of the level of GTP binding when the compound is present at l ⁇ M or less.
  • GTPase activity is measured by incubating the membranes containing a TIR or T2R polypeptide with . ⁇ 32 P-GTP. Active GTPase will release the label as inorganic phosphate, which is detected by separation of free inorganic phosphate in a 5% suspension of activated charcoal in 20 mM HsPO , followed by scintillation counting. Controls include assays using membranes isolated from cells not expressing TlR or T2R (mock-transfected), in order to exclude possible non-specific effects of the candidate compound.
  • membrane samples are incubated with and without the modulator, followed by the GTPase assay.
  • a change (increase or decrease) of 10% or more in the level of GTP bindmg or GTPase activity relative to samples without modulator is indicative of TIR or T2R modulation by a candidate modulator.
  • aequorin assay takes advantage of the responsiveness of mitochondrial apoaequorin to intracellular calcium release induced by the activation of GPCRs (Stables et al, Anal. Biochem. 252:115-126 (1997); Detheux et al., 2000, J. Exp. Med., 192 1501-1508 (2000) (131-132); both of which are incorporated herein by reference). Briefly, TlR or T2R-expressing clones are transfected to coexpress mitochondrial apoaequorin and G ⁇ i6.
  • Cells are incubated with 5 ⁇ M Coelenterazine H (Molecular Probes) for 4 hours at room temperature, washed in DMEM-F12 culture medium and resuspended at a concentration of 0.5.times.l0.sup.6 cells/ml. Cells are then mixed with test agonist molecules and light emission by the aequorin is recorded with a lummometer for 30 seconds. Results are expressed as Relative Light Units (RLU). Controls include assays using membranes isolated from cells not expressing TIR or T2R (mock transfected), in order to exclude possible non-specific effects of the candidate compound.
  • RLU Relative Light Units
  • Aequorin activity or intracellular calcium levels are "changed” if light intensity increases or decreases by 10% or more in a sample of cells, expressing a TlR or T2R polypeptide and treated with a candidate modulator, relative to a sample of cells expressing the TlR or T2R polypeptide but not treated with the candidate modulator or relative to a sample of cells not expressing the TlR or T2R polypeptide (mock-transfected cells) but treated with the candidate modulator.
  • Adenylate Cyclase Assay [00160] Assays for adenylate cyclase activity are described by Kenimer & Nirenberg, Mol. Pharmacol. 20: 585-591 (1981) (133). That assay is a modification of the assay taught by Solomon et al., 1974, Anal. Biochem. 58: 541- 548 (1974) (134), also incorporated herein by reference.
  • lOO ⁇ l reactions contain 50 mM Tris-Hcl (pH 7.5), 5 mM MgCb, 20 mM creatine phosphate (disodium salt), 10 units (71 . ⁇ g of protein) of creatine phosphokinase, 1 mM ⁇ -32P (tetrasodium salt, 2 ⁇ Ci), 0.5 mM cyclic AMP, G" 3H -labeled cyclic AMP (approximately 10,000 cpm), 0.5 mM Ro20-1724, 0.25% ethanol, and 50-200 ⁇ g of protein homogenate to be tested (i.e., homogenate from cells expressing or not expressing a TlR or T2R polypeptide, treated or not treated with a candidate modulator).
  • protein homogenate to be tested i.e., homogenate from cells expressing or not expressing a TlR or T2R polypeptide, treated or not treated with a candidate modulator.
  • Reaction mixtures are generally incubated at 37°C. for 6 minutes. Following incubation, reaction mixtures are deproteinized by the addition of 0.9 ml of cold 6% trichloroacetic acid. Tubes are centrifuged at 1800xg for 20 minutes and each supernatant solution is added to a Dowex AG50W-X4 column. The cAMP fraction from the column is eluted with 4 ml of 0.1 mM imidazole-HCl (pH 7.5) into a counting vial. Assays should be performed in triplicate. Control reactions should also be performed using protein homogenate from cells that do not express a TlR or T2R polypeptide.
  • adenylate cyclase activity is "changed” if it increases or decreases by 10% or more in a sample taken from cells treated with a candidate modulator of TIR or T2R activity, relative to a similar sample of cells not treated with the candidate modulator or relative to a sample of cells not expressing the TIR or T2R polypeptide (mock-transfected cells) but treated with the candidate modulator.
  • Intracellular or extracellular cAMP is measured using a cAMP radioimmunoassay (RIA) or cAMP binding protein according to methods widely known in the art. For example, Horton & Baxendale, Methods Mol. Biol 41: 91- 105 (1995) (135), which is incorporated herein by reference, describes an RIA for cAMP.
  • RIA radioimmunoassay
  • kits for the measurement of cAMP are commercially available, such as the High Efficiency Fluorescence Polarization-based homogeneous assay marketed by LJL Biosystems and NEN Life Science Products. Control reactions should be performed using extracts of mock- transfected cells to exclude possible non-specific effects of some candidate modulators.
  • the level of cAMP is "changed” if the level of cAMP detected in cells, expressing a TIR or T2R polypeptide and treated with a candidate modulator of TIR or T2R activity (or in extracts of such cells), using the RIA-based assay of Horton & Baxendale, 1995 (135), increases or decreases by at least 10% relative to the cAMP level in similar cells not treated with the candidate modulator.
  • Triphosphate Levels [00167] Receptors that activate the breakdown of phospholipids can be monitored for changes due to the activity of known or suspected modulators of TlR or T2R by monitoring phospholipid breakdown, and the resulting production of second messengers DAG and or inositol triphosphate (IP3). Methods of detecting each of these are described in Phospholipid Signalling Protocols, edited by Ian M. Bird. Totowa, N.J., Humana Press, (1998) (136), which is incorporated herein by reference. See also Rudolph et al., J. Biol. Chem. 274: 11824-11831 (1999) (137), which also describes an assay for phosphatidylinositol breakdown.
  • Assays should be performed using cells or extracts of cells expressing TlR or T2R, treated or not treated or without a candidate modulator. Control reactions should be performed using mock-transfected cells, or extracts from them in order to exclude possible non-specific effects of some candidate modulators.
  • phosphatidylinositol breakdown, and diacylglycerol and/or inositol triphosphate levels are "changed” if they increase or decrease by at least 10% in a sample from cells expressing a TIR or T2R polypeptide and treated with a candidate modulator, relative to the level observed in a sample from cells expressing a TIR or T2R polypeptide that is not treated with the candidate modulator.
  • PKC Protein Kinase C
  • I CAM I intracellular adhesion molecule I
  • Assays designed to detect increases in gene products induced by PKC can be used to monitor PKC activation and thereby receptor activity.
  • the activity of receptors that signal via PKC can be monitored through the use of reporter gene constructs driven by the control sequences of genes activated by PKC activation. This type of reporter gene-based assay is discussed in more detail below.
  • the substrate for the assay is the peptide AC-FKKSFKL-NH2, derived from the myristoylated alanine-rich protein kinase C substrate protein (MARCKS).
  • the K m of the enzyme for this peptide is approximately 50 ⁇ M.
  • Other basic, protein kinase C-selective peptides known in the art can also be used, at a concentration of at least 2-3 times their K m .
  • Cofactors required for the assay include calcium, magnesium, ATP, phosphatidylserine and diacylglycerol.
  • the assay can be performed to determine the amount of PKC present (activating conditions) or the amount of active PKC present (non- activating conditions).
  • activating conditions the amount of PKC present
  • non- activating conditions will be used, such that the PKC, that is active in the sample when it is isolated, is measured, rather than measuring the PKC that can be activated.
  • calcium is omitted from the assay in favor of EGTA.
  • the assay is performed in a mixture containing 20 mM HEPES, pH 8.
  • Assays are performed on extracts from cells expressing a TIR or T2R polypeptide, treated or not treated with a candidate modulator. Control reactions should be performed using mock-transfected cells, or extracts from them in order to exclude possible non-specific effects of some candidate modulators.
  • PKC activity is "changed" by a candidate modulator when the units of PKC measured by either assay described above increase or decrease by at least 10%, in extracts from cells expressing TlR or T2R and treated with a candidate modulator, relative to a reaction performed on a similar sample from cells not treated with a candidate modulator.
  • MAP Kinase assays have already been described supra. MAP kinase activity can be assayed using any of several kits available commercially, for example, the p38 MAP Kinase assay kit sold by New England Biolabs (Cat # 9820) or the FlashPlateTM MAP Kinase assays sold by Perkin-Elmer Life Sciences.
  • MAP Kinase activity is "changed” if the level of activity is increased or decreased by 10% or more in a sample from cells, expressing a TIR or T2R polypeptide, treated with a candidate modulator relative to MAP kinase activity in a sample from similar cells not treated with the candidate modulator.
  • Direct assays for tyrosine kinase activity using known synthetic or natural tyrosine kinase substrates and labelled phosphate are well known, as are similar assays for other types of kinases (e.g., Ser/Thr kinases).
  • Kinase assays can be performed with both purified kinases and crude extracts prepared from cells expressing a TlR or T2R polypeptide, treated with or without a candidate modulator. Control reactions should be performed using mock-transfected cells, or extracts from them in order to exclude possible non-specific effects of some candidate modulators.
  • Substrates can be either full-length protein or synthetic peptides representing the substrate. Pinna & Ruzzene (Biochem. Biophys. Acta 1314: 191-225 (1996) (139)) list a number of phosphorylation substrate sites useful for detecting kinase activities. A number of kinase substrate peptides are commercially available.
  • RRLIEDAEYAARG a substrate for many receptor and nonreceptor tyrosine kinases. Because the assay described below requires binding of peptide substrates to filters, the peptide substrates should have a net positive charge to facilitate binding. Generally, peptide substrates should have at least 2 basic residues and a free amino terminus. Reactions generally use a peptide concentration of 0.7-1.5 mM.
  • Assays are generally carried out in a 25 ⁇ l volume comprising 5 .mu.l of 5X kinase buffer (5 mg/mL BSA, 150 mM Tris-Cl (pH 7.5), 100 mM MgCk; depending upon the exact kinase assayed for, M11CI2 can be used in place of or in addition to the MgCk), 5 .mu.l of 1.0 mM ATP (0.2 mM final concentration), ⁇ 32 .
  • 5X kinase buffer 5 mg/mL BSA, 150 mM Tris-Cl (pH 7.5), 100 mM MgCk; depending upon the exact kinase assayed for, M11CI2 can be used in place of or in addition to the MgCk
  • 5 .mu.l of 1.0 mM ATP (0.2 mM final concentration) ⁇ 32 .
  • ATP 100-500 cpm/pmol
  • 3 ⁇ l of 10 mM peptide substrate 1.2 mM final concentration
  • cell extract containing kinase to be tested cell extracts used for kinase assays should contain a phosphatase inhibitor (e.g. 0.1-1 mM sodium orthovanadate)), and H2O to 25 ⁇ l. Reactions are performed at 30°C, and are initiated by the addition of the cell extract.
  • phosphatase inhibitor e.g. 0.1-1 mM sodium orthovanadate
  • the specific activity of ATP in the kinase reaction (e.g., in cpm/pmol) is determined by spotting a small sample (2-5 ⁇ l) of the reaction onto a P81 filter circle and counting directly, without washing. Counts per minute obtained in the kinase reaction (minus blank) are then divided by the specific activity to determine the moles of phosphate transferred in the reaction.
  • Tyrosine kinase activity is "changed” if the level of kinase activity is increased or decreased by 10% or more in a sample from cells, expressing a TIR or T2R polypeptide, treated with a candidate modulator relative to kinase activity in a sample from similar cells not treated with the candidate modulator.
  • Transcriptional Reporters for Downstream Pathway Activation [00186]
  • the intracellular signal initiated by binding of an agonist to a receptor e.g., TIR or T2R, sets in motion a cascade of intracellular events, the ultimate consequence of which is a rapid and detectable change in the transcription or translation of one or more genes.
  • the activity of the receptor can therefore be monitored by detecting the expression of a reporter gene driven by control sequences responsive to TIR or T2R activation.
  • promoter refers to the transcriptional control elements necessary for receptor-mediated regulation of gene expression, including not only the basal promoter, but also any enhancers or transcription- factor binding sites necessary for receptor-regulated expression.
  • Reporter genes such as luciferase, CAT, GFP, ⁇ -lactamase or ⁇ - galactosidase are well known in the art, as are assays for the detection of their products.
  • Genes particularly well suited for monitoring receptor activity are the "immediate early" genes, which are rapidly induced, generally within minutes of contact between the receptor and the effector protein or ligand.
  • the induction of immediate early gene transcription does not require the synthesis of new regulatory proteins.
  • characteristics of preferred genes useful for making reporter constructs include: low or undetectable expression in quiescent cells; induction that is transient and independent of new protein synthesis; subsequent shut-off of transcription requires new protein synthesis; and mRNAs transcribed from these genes have a short half-life. It is preferred, but not necessary that a transcriptional control element have all of these properties for it to be useful.
  • c-fos proto-oncogene An example of a gene that is responsive to a number of different stimuli is the c-fos proto-oncogene.
  • the c-fos gene is activated in a protein- synthesis-independent manner by growth factors, hormones, differentiation- specific agents, stress, and other known inducers of cell surface proteins.
  • the induction of c-fos expression is extremely rapid, often occurring within minutes of receptor stimulation. This characteristic makes the c-fos regulatory regions particularly attractive for use as a reporter of receptor activation.
  • the c-fos regulatory elements include (see, Verma et al., Cell 51: 513- 514) (1987) (140): a TATA box that is required for transcription initiation; two upstream elements for basal transcription, and an enhancer, which includes an element with dyad symmetry and which is required for induction by TPA, serum,
  • EGF EGF
  • PMA PMA
  • the 20 bp c-fos transcriptional enhancer element located between -317 and -298 bp upstream from the c-fos mRNA cap site, is essential for serum induction in serum starved NIH 3T3 cells.
  • One of the two upstream elements is located at -63 to -57 and it resembles the consensus sequence for cAMP regulation.
  • the transcription factor CREB (cyclic AMP responsive element binding protein) is, as the name implies, responsive to levels of intracellular cAMP. Therefore, the activation of a receptor that signals via modulation of cAMP levels can be monitored by detecting either the binding of the transcription factor, or the expression of a reporter gene linked to a CREB-binding element (termed the CRE, or cAMP response element).
  • the DNA sequence of the CRE is TGACGTCA.
  • VIP vasoactive intestinal peptide
  • cAMP responsive Fink et al., 1988, Proc. Natl. Acad. Sci. 85:6662-6666
  • somatostatin gene promoter cAMP responsive; Montminy et al, Proc. Natl. Acad. Sci.
  • transcriptional control elements that are responsive to changes in GPCR activity include, but arc not limited to those responsive to the AP-1 transcription factor and those responsive to NF-KB activity.
  • the consensus AP-1 binding site is the palindrome TGA(C/G)TCA (Lee et al., Nature 325: 368-372 (1987) (146); Lee et al., Cell 49: 741-752 (1987) (147)).
  • the AP-1 site is also responsible for mediating induction by tumor promoters such as the phorbol ester 12-O-tetradecanoylphorbol-.beta.-acetate (TPA), and are therefore sometimes also referred to as a TRE, for TPA-response element.
  • TPA phorbol ester 12-O-tetradecanoylphorbol-.beta.-acetate
  • TRE for TPA-response element.
  • AP-1 activates numerous genes that are involved in the early response of cells to growth stimuli. Examples
  • Fos-related antigens 1 and 2, 1 K ⁇ , ornithine decarboxylase, and annexins
  • the NF-KB binding element has the consensus sequence GGGGACTTTCC.
  • a large number of genes have been identified as NF-KB responsive, and their control elements can be linked to a reporter gene to monitor GPCR activity.
  • a small sample of the genes responsive to NF-KB includes those encoding IL-l ⁇ . (Hiscott et al., Mol. Cell. Biol. 13:6231-6240 (1993) (148)), TNF- ⁇ (Shakhov et al., J. Exp. Med. Ill: 35-47 (1990) (149)), CCR5 (Liu et al., AIDS Res. Hum.
  • NF-KB-responsive reporters are also known in the art or can be readily made by one of skill in the art using, for example, synthetic NF-KB elements and a minimal promoter, or using the NF- KB-responsive sequences of a gene known to be subject to NF-KB regulation. Further, NF-KB responsive reporter constructs are commercially available e.g., from CLONTECH.
  • the cells are left untreated, exposed to candidate modulators, and expression of the reporter is measured.
  • An increase of at least 50% in reporter expression in the presence of a candidate modulator indicates that the candidate is a modulator of TlR or T2R activity.
  • An agonist will induce at least as many, and preferably the same amount or more of reporter expression than buffer alone.
  • This approach can also be used to screen for inverse agonists where cells express a TIR or T2R polypeptide at levels such that there is an elevated basal activity of the reporter.
  • a decrease in reporter activity of 10% or more in the presence of a candidate modulator, relative to its absence, indicates that the compound is an inverse agonist.
  • the cells expressing TlR or T2R and carrying the reporter construct are contacted in the presence and absence of a candidate modulator.
  • Controls for transcription assays include cells not expressing TlR or T2R but carrying the reporter construct, as well as cells with a promote less reporter construct.
  • Compounds that are identified as modulators of TlR or T2R- regulated transcription should also be analyzed to determine whether they affect transcription driven by other regulatory sequences and by other receptors, in order to determine the specificity and spectrum of their activity.
  • the transcriptional reporter assay and most cell-based assays, are well suited for screening expression libraries for proteins for those that modulate TIR or T2R activity.
  • the libraries can be, for example, cDNA libraries from natural sources, e.g., plants, animals, bacteria, etc., or they can be libraries expressing randomly or systematically mutated variants of one or more polypeptides.
  • Genomic libraries in viral vectors can also be used to express the mRNA content , of one cell or tissue, in the different libraries used for screening of TIR or T2R.
  • Cells of the invention are labelled for 24 hours with 10 ⁇ Ci ml 3 H] inositol in inositol free DMEM containing 5% FCS, antibiotics, amphotericin, sodium pyruvate and 400 ⁇ g/ml G418.
  • Cells are incubated for 2 h in Krebs- Ringer Hepes (KRH) buffer of the following composition (124 mM NaCI, 5 mM KCl, 1.25 mM MgSO , 1.45 mM CaCl 2 , 1.25 mM KH 2 PO 4 , 25 mM Hepes (pH:7.4) and 8 mM glucose).
  • KRH Krebs- Ringer Hepes
  • the cells are then challenged with various nucleotides for 30 s.
  • TIR or T2R Assay provides for an assay for detecting the activity of a receptor of the invention in a sample.
  • TIR or T2R activity can be measured in a sample comprising a cell or a cell membrane that expresses TlR or T2R.
  • the assay is performed by incubating the sample in the presence or absence of a modulator and carrying out a second messenger assay, as described above. The results of the second messenger assay performed in the presence or absence of the activator are compared to determine if the TlR or T2R receptor is active.
  • any of the assays of receptor activity including but not limited to the GTP-binding, GTPase, adenylate cyclase, cAMP, phospholipid-breakdown, diacylglycerol, inositol triphosphate, arachidonic acid release (see below), PKC, kinase and transcriptional reporter assays, can be used to determine the presence of an agent in a sample, e.g., a tissue sample, that affects the activity of the TIR or T2R receptor molecule. To do so, TIR or T2R polypeptide is assayed for activity in the presence and absence of the sample or an extract of the sample.
  • TlR or T2R activity in the presence of the sample or extract relative to the absence of the sample indicates that the sample contains an agonist of the receptor activity.
  • a decrease in receptor activity in the presence of an agonist and the sample, relative to receptor activity in the absence thereof, indicates that the sample contains an antagonist of TIR or T2R activity.
  • the amount of increase or decrease in measured activity necessary for a sample to be said to contain a modulator depends upon the type of assay used.
  • a 10% or greater change (increase or decrease) relative to an assay performed in the absence of a sample indicates the presence of a modulator in the sample.
  • the transcriptional reporter assay in which at least a two-fold increase or 10% decrease in signal is necessary for a sample to be said to contain a modulator.
  • an agonist stimulates at least 50%, and preferably 75% or 100% or more, e.g., 2-fold, 5-fold, 10-fold or greater receptor activation.
  • Other functional assays include, for example, microphysiometer or biosensor assays (see Hafner, 2000, Biosens. Bioelectron. 15: 149-158) (2000) (155)).
  • cell-based assays e.g., MAPK and cAMP assay methods exemplified, enable the detection of robust activation of bitter taste receptors (mT2R05) and hT2R04 as well as the sweet receptor (T1R2/T1R3) and umami receptor (T1R1/T1R3).
  • MAPK and cAMP assay methods exemplified, enable the detection of robust activation of bitter taste receptors (mT2R05) and hT2R04 as well as the sweet receptor (T1R2/T1R3) and umami receptor (T1R1/T1R3).
  • the results obtained indicate that the responses obtained are receptor-dependent and receptor-specific.
  • the parental cell lines HEK293 or HEK293T-G ⁇ s do not exhibit comparable activation of MAPK or a reduction in cAMP (See Figures 1-7) when stimulated with the same agonists.
  • the subject MAPK assays are exemplified by the above-described antibody-based methods for detecting MAPK activation. As noted supra, however, the invention encompasses any suitable assay system for detecting activated MAPK. (71) Vaster et al., Biochem J. 350:717-22 (2000), incorporated by reference in its entirety herein, describes a phosphospecific cell-based ELISA for detecting p42/p44 MAPK, p38MAPK, protein kinase B and cAMP response- element binding protein. This assay, referred to as "PACE”, (phosphospecific antibody cell-based ELISA) detects activated MAPK without the use of radioactive labels, and can use adherent cells or cells in suspension.
  • PACE phosphospecific antibody cell-based ELISA
  • the detection of MAPK activation can be effected by the use of proximity assays (AlphaScreenTM) from Packard or by use of High Content Screen System (ERK MAPK Activation HitKitTM ) from Cellomics. These assays or other available MAPK assays, can be used as part of a high throughput screening platform for identifying bitter, sweet and umami receptor agonists and antagonists.
  • proximity assays AlphaScreenTM
  • ERK MAPK Activation HitKitTM High Content Screen System
  • cAMP accumulation is measured by an immunofluorescence assay as described in the examples.
  • the subject invention embraces the use of any suitable means for detecting cAMP levels.
  • Such methods include the detection of cAMP using anti- cAMP antibodies in an ELISA-based format, or by second messenger reporter system assays.
  • Promoters on genes drive the expression of the proteins that a particular gene encodes.
  • Cyclic AMP drives gene expression by promoting the binding of a cAMP-responsive DNA binding protein or transcription factor (CREB) that then binds to the promoter at specific sites called cAMP response elements and drives the expression of the gene.
  • CREB cAMP-responsive DNA binding protein or transcription factor
  • reporter systems can be constructed which have a promoter containing multiple cAMP response elements before a reporter gene, e.g., beta-galactosidose or luciferase.
  • a constitutively activated Gi linked receptor causes a reduction in cAMP that results in inhibition of the gene expression and reduced expression of the reporter gene.
  • the reporter protein can be detected using standard biochemical assays.
  • the present invention relates to the discovery that TlRs and T2Rs functionally couple to G proteins other than promiscuous G proteins such as G ⁇ is or gustducin.
  • the invention involves the discovery that TlRs and T2Rs functionally couple to Gi proteins and use G ⁇ i to transmit signals to downstream effectors, e.g., adenylyl cyclase and MAP Kinase.
  • Gs stimulates the enzyme adenylyl cyclase .
  • Gi and G z and Go
  • Adenylyl cyclase catalyzes the conversion of ATP to cAMP.
  • constitutively activated GPCRs that couple Gi (or G z and Go) protein associated with a decrease in cellular levels of cAMP. See, generally, “Indirect Mechanisms of Synoptic Transmission,” Chapter 8, From Neuron to Brain (3rd Edition), Nichols, J.G. et al etds., Sinaver Associates, Inc. (1992).
  • assays that detect cAMP can be utilized to determine if a compound is e.g., an inverse agonist to the receptor (i.e., such a compound would decrease the levels of cAMP):
  • a variety of approaches can be used to measure cAMP, e.g., anti-cAMP antibodies in an ELISA method, or the second messenger reporter system assays described supra.
  • a Gi protein coupled receptor is known to inhibit adenylyl cyclase, resulting in a decrease in cAMP production.
  • Another effective technique for measuring the decrease in production of cAMP as an indication of constitutive activation of a receptor that predominantly couples Gi upon activation can be accomplished by co-transfecting a signal enhancer, e.g., a non- endogenous, constitutively activated receptor that predominantly couples with Gs upon activation with the Gi linked GPCR, i.e., a TIR or T2R.
  • constitutive activation of a G s coupled receptor can be determined based upon an increase in production of cAMP.
  • TlR or T2R modulators using such a cAMP assay can then be accomplished with two provisos: first, relative to the Gi coupled target receptor (TIR or T2R), "opposite" effects will result, i.e., an inverse agonist of the Gi coupled target receptor will decrease this signal; second candidate modulators that are identified using this approach should be assessed independently to ensure that these compounds do not target the signal enhancing receptor (this can be accomplished prior to or after screening against co-transfected receptor).
  • TIR or T2R Gi coupled target receptor
  • Particular signal substances that use cAMP as a second messenger include by way of example calcitonin, chorionic gonadotropin, corticotrophin, epinephrine, follicle- stimulating homone, glucagon, leutenizing hormone, lipotropin, melanocyte-stimulating hormone, nor epinephrine, parathyroid hormone (PTH), thyroid-stimulating hormone and vasopressin.
  • cycloheximide a bitter compound, specifically activates ERKl/2 mitogen-activated kinases in cells expressing the mouse bitter receptor mT2R5 and the rat bitter receptors rT2R9. Consistent with the foregoing, activation of ERKl/2 is totally abolished upon treatment with pertussis toxin indicating that these receptors couple to ERKl/2 activation through G ⁇ i. Also in agreement with these observations, cycloheximide inhibits the forskolin-induced cAMP accumulation in mT2R5-expressing cells by 70%.
  • natural and artificial sweeteners such as sucrose, D-tryptophan, saccharin and cyclamate (known activates of T1R2/T1R3 sweet receptors) activate ERKl/2 in cells expressing the human sweet receptor hTlR2/hTlR3.
  • monosodium glutamate exclusively activates ERKl/2 in cells expressing the human umami receptor hTlRl/hTlR3 and the effect is greatly enhanced by the presence of inosine monophosphate. Again, consistent with Gi coupling, these responses are prevented by treatment with pertussis toxin.
  • sweeteners including cyclamate, aspartame, saccharin, and monellin significantly mhibit the forskolin-induced cAMP accumulation in hTlR2/hTlR3-expressing cells by 50-70%.
  • Monosodium glutamate also decreases basal levels of cAMP in hTlRl/hTlR3-expressing cells by 50%.
  • G protein subunit, ⁇ -gustducin (78) (McLaughlin et al., Nature 357:563-569 (1992)), produces mice that are defective in detection of bitter and sweet substances (17).
  • the visual G-protein ⁇ -transducin is also expressed in taste tissue (74, 75) (Ruiz-Avila et al., Nature 376:80-85 (1995); McLaughlin et al.,
  • G ⁇ s , G ⁇ is, G ⁇ u-i,, G ⁇ u-2, G ⁇ i-s and G ⁇ q has been detected in taste tissues using RT-PCR (15, 25).
  • G ⁇ n-2 can also be detected by in situ hybridization (25, 26) and immunostaining (25) in TRCs and a study by Hoon et al., (32) reported that Gi proteins are expressed in almost all TRCs.
  • G ⁇ u-2 positive cells are thought to be larger in number than G ⁇ -gustducin-positive cells in rat circumvallate papillae (Kusakabe et al., Chem. Senses 25(5):525-31 (2000) (25)).
  • mice retain residual responsiveness to bitter and sweet stimuli (Wong et al., Nature 381:796-800 (1996); He et al, Chem Senses 27(8): 719-27 (2002); Ruiz-Avila et al, PNC Natl Acad Sci, USA 98(15): 541-551 (2001) (17, 27, 28)) suggesting that another G protein may complement ⁇ -gustducin functions in TRCs.
  • the present inventors have studied coupling of receptors for bitter, sweet and umami taste to classical GPCR-linked signaling pathways in HEK293 cells, and the results obtained surprisingly demonstrate that these taste receptors can effectively couple to G ⁇ i-dependent activation of mitogen activated protein (MAP) kinases ERKl and ERK2 (ERKl/2) and G ⁇ i-dependent inhibition of cAMP accumulation. Also, these results further surprisingly indicate that the sweet receptor does not couple to G s stimulation and accumulation of cAMP. Functional coupling to G ⁇ i may explain, in part, the observations that bitter-tasting substances and MSG decrease the level of cyclic nucleotides in TRCs. Moreover, these results suggest that G ⁇ i can functionally complement ⁇ -gustducin functions in TRCs.
  • MAP mitogen activated protein
  • the present invention provides cell-based assay methods that rely on the discovery that TlRs and T2Rs functionally couple to Gi proteins e.g., G ⁇ i and transmit signals to downstream effectors, e.g., cAMP, MAP Kinase, and adenylyl cyclase that enable the identification of modulators, e.g., agonists, antagonists, inverse agonists enhancers of a TlR or T2R polypeptide.
  • modulators e.g., agonists, antagonists, inverse agonists enhancers of a TlR or T2R polypeptide.
  • the T2R modulators of the invention are useful for altering taste perception, for example to induce, suppress or enhance bitter taste perception in a subject.
  • the T1R2/T1R3 modulators are useful for modulating sweet taste, e.g., by enhancing the taste of another sweet tasting compound such as saccharin.
  • the T1R1/T1R3 modulators identified according to the invention are useful for modulating umami taste, e.g., by enhancing the taste of a umami compound such as monosodium glutamate.
  • a composition that is administered to alter taste perception in a subject will comprise an effective amount of a TlR or T2R modulator (agonist, antagonist, or enhancer).
  • a TlR or T2R activator or modulator can comprise any substance e.g., small molecule, peptide, protein, carbohydrate, oligosaccharide, glycoprotein, amino acid derivative, and the like.
  • compounds will be identified by screening libraries of potential taste modulatory compounds, which may be comprised of synthetic or naturally occurring compounds. The library may be random or may comprise compounds having related structures or are structures or substitutions.
  • TlR or T2R modulators identified as disclosed herein can be used to prepare compositions suitable for oral use, including but not limited to food, beverages, oral washes, dentifrices, cosmetics, and pharmaceuticals.
  • TlR or T2R modulators can also be used as additives to alter the sweet, umami or bitter taste of a compound that is of palatable but undesirable for oral use, for example compounds comprised in household cleansers, poisons, etc. Such modulators will alter bitter, sweet or umami tasting compounds contained therein.
  • representative foods having an undesirable or bitter taste include, but are not limited to, citrus fruits such as grapefruit, orange, and lemon; vegetables such as tomato, pimento, celery, melon, carrot, potato, and asparagus; seasoning or flavoring materials such as flavor, sauces, soy sauce, and red pepper; foods originating from soybean; emulsion foods such as cream, dressing, mayonnaise, and margarine; processed marine products such as fish meat, ground fish meat, and fish eggs; nuts such as peanuts; fermented foods such as fermented soybean; meats and processed meats; pickles; noodles; soups including powdery soups; dairy products such as cheese; breads and cakes; confectioneries such as candies, chewing gum, and chocolate; and specifically prepared foods for health.
  • citrus fruits such as grapefruit, orange, and lemon
  • vegetables such as tomato, pimento, celery, melon, carrot, potato, and asparagus
  • seasoning or flavoring materials such as flavor, sauces, soy sauce, and red pepper
  • foods originating from soybean emulsion
  • Cosmetics eliciting bitter taste include but are not limited to those compositions that include surfactants such as sodium alkyl sulfate and sodium monoalkyl phosphate; fragrances such as menthol, linalool, phenylethyl alcohol, ethyl propionate, geraniol, linalyl acetate and benzyl acetate; antimicrobials such as methyl paraben, propyl paraben and butyl paraben; humectants such as lactic acid and sodium lactate; alcohol-denaturating agents such as sucrose octaacetate and brucine; and astringents such as aluminum lactate.
  • surfactants such as sodium alkyl sulfate and sodium monoalkyl phosphate
  • fragrances such as menthol, linalool, phenylethyl alcohol, ethyl propionate, geraniol, linalyl acetate and benzyl acetate
  • Representative pharmaceuticals having a bitter taste include acetaminophen, terfenadine, guaifenesin, trimethoprim, prednisolone, ibuprofen, prednisolone sodium phosphate, methacholine, pseudoephedrine hydrochloride, phenothiazine, chlorpromazine, diphenylhydantoin, caffeine, morphine, demerol, codeine, lomotil, lidocaine, salicylic acid, sulfonamides, chloroquine, a vitamin preparation, minerals and penicillins, neostigmine, epinephrine, albuterol, diphenhydr amine, chlorpheniramine maleate, chlordiazepoxide, amitriptyline, barbiturates, diphenylhydantoin, caffeine, morphine, demerol.
  • Representative sweeteners which may be modulated by compounds according to the invention include xylitol, sorbitol, saccharin, sucrose, glucose, fructose, cyclamate, aspartame, monellin, and the like, and derivatives thereof.
  • umami compounds the taste which may be modulated according to the invention include L-glutamate, L-asparate, monosodium glutamate, derivatives thereof, compounds containing and the like.
  • taste modulators can also be administered as part of prepared food, beverage, oral wash, dentifrice, cosmetic, or drug.
  • a TIR or T2R modulator can be admixed with a compound, the taste of which is to be modulated in amount comprising about 0.001 % to about 10% by weight, preferably from about 0.01% to about 8% by weight, more preferably from about 0.1 % to about 5% by weight, and most preferably from about 0.5% to about 2% by weight.
  • Suitable formulations include solutions, extracts, elixirs, spirits, syrups, suspensions, powders, granules, capsules, pellets, tablets, and aerosols.
  • a formulation can include a pharmaceutically acceptable carrier, a suspending agent, a solubilizer, a thickening agent, a stabilizer, a preservative, a flavor, a colorant, a sweetener, a perfume, or a combination thereof.
  • TlR or T2R modulators and compositions can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
  • Administration [00236] TIR or T2R modulators can be administered directly to a subject for modulation of taste perception. Preferably, a modulator of the invention is administered orally or nasally.
  • an effective amount of a TIR or T2R modulator is administered to a subject.
  • the term "effective amount” refers to an amount of a composition sufficient to modulate TlR or T2R activation and/or to modulate taste perception, e.g., bitter, sweet or umami taste perception.
  • An effective amount can be varied so as to administer an amount of an TlR or T2R modulator that is effective to achieve the desired taste perception.
  • the selected dosage level will depend upon a variety of factors including the activity of the TlR or T2R modulator, formulation, combination with other compositions (e.g., food, drugs, etc.), the intended use (e.g., as a food additive, dentifrice, etc.), and the physical condition and prior medical history of the subject being treated.
  • An effective amount or dose can be readily determined using in vivo assays of taste perception as are known in the art. Representative methods for assaying taste perception are described infra.
  • Sweeteners, agonists and toxins Sucrose, aspartame, cyclamate, monellin, monosodium glutamate, inosine monophosphate, isoproterenol, epidermal growth factor, denatonium benzoate, quinine sulfate, cycloheximide, rolipram and forskolin were from Sigma (St-Louis, MO). Pertussis toxin (PTX) was from List Biological Laboratories (Campbell, CA).
  • hTlRl/hTlR3 umami taste receptor line
  • Vectors were prepared using the GeneSwitch inducible system (Invitrogen, Carlsbad, CA).
  • hTlRl and hTlR3 vectors were prepared by cloning receptor cDNA into pGene/V5-His A at EcoRI/Not I sites.
  • a modified pSwitch vector was also prepared by replacing the hygromycin ⁇ resistance gene with the puromycin resistance gene.
  • hTlRl hTlR3
  • puromycin resistance was co-transfected into HEK293 cells stably expressing G ⁇ is (Aurora Biosciences, San Diego, (80) Chandraskekar et al, Cell 100(6): 703-11 (2000).
  • hTlRl/hTlR3 stable cell lines were selected and maintained in high-glucose DMEM media containing lOO ⁇ g/mL zeocin, 0.5 ⁇ g/mL puromycin, 2mM GIutaMAX 1, 10% dialyzed fetal bovine serum, 3 ⁇ g/mL blasticidin and penicillin/streptomyocin.
  • Matrigel Becton-
  • hTlRl and hTlR3 were induced by treatment of cells with 6xlO- u M mifepristone for 48 hours prior to experiments. Clones were tested and selected for mifepristone-induced responsiveness to MSG/IMP using calcium-imaging experiments (data not shown). The clone used in this study did not show any functional expression of hTlRl/R3 without induction (data not shown).
  • HEK293 cells were transfected with 5 ⁇ g of linearized Rho-mT2R5 plasmid (80) Chandraskekar et al (2000) in pEAKlO (Edge biosystems) using the Transit transfection reagent (Panvera). Cells were selected in the presence of 0.5 ⁇ g/ml puromycin, clones were isolated, expanded and analyzed by fluorescence- activated cell sorting for the presence of Rho tag immunoreactivity at the cell surface using a monoclonal antibody; raised against the first 40 amino acids of rhodopsin (80, 81) (Chandrashekar et al (2000); Adamus et al., Vision Res. 31(1): 17-31 (1991)).
  • EXAMPLE 1 MAP Kinase Assays [00245] Transient transfection of HEK293 cells for ERK112 assay. Subclonfluent HEK293 cells in 10cm dishes were transfected with 4 ⁇ g of Rho- rT2R9 plasmid (Chandrashekar et al (2000); Bute et al. 3 J. Receptor Signal Transduct. Res. 20(2-3): 153-166 (2000)) pEAKlO (Edge Biosystems, Gaithersburg, MD (80, 82)) and 2 ⁇ g pUC-18 as a carrier DNA using the Transit transfection reagent (Panvera). 24 hours later, cells were harvested using Hank's balanced salt solution without calcium or magnesium and containing 1 mM EDTA (HBSS/EDTA), and plated into 6 well plates. ERKl/2 assay was performed 48 hours post-transfection.
  • Rho- rT2R9 plasmid Chandrash
  • Lysis buffer containing 150mM NaCI, 50mM TrisHEl pH 8., 0.25% sodium deoxycholate, 1 % igepal (NP- 40), 2mM sodium orthovanadate, ImM sodium fluoride, and protease inhibitors were then added and cells were scraped off the plates. Lysates were frozen immediately in liquid nitrogen and kept at -80°C until further analysis.
  • Lysate protein concentration was determined using the Bradford method (Amresco, Solon, OH).
  • Cell lysate proteins 22 FLg/lane) were resolved by SDS-PAGE using 4-20% Tris-glycine gels (Invitrogen, Carlsbad, CA). Following electrophoresis, proteins were transferred to nitrocellulose membranes that were subsequently blocked with 5% fat-free milk in Tris-buffer saline containing 0.2% tween-20 (TBST).
  • TST Tris-buffer saline containing 0.2% tween-20
  • Membranes were immunoblotted with phospho-p44/42 MAPK monoclonal antibody (Cell Signaling Technology, Beverly, MA) diluted 1:1000 in 5% milk/TBST overnight at 4°C.
  • Membranes were stripped of phospho-specific antibodies using 0.2 M glycine pH 2.5 and re-blotted with p44/42 polyclonal antibodies (Cell Signaling Technology, Beverly, MA) diluted 1:1000 in 5% milk/TBST overnight at 4°C. Secondary antibody was HRP-linked anti-rabbit IgG diluted 1:2000 in 5% milk/TBST.
  • cAMP content of cells was determined by a commercially-available chemiluminescent immunoassay kit (Applied Biosystems, Foster City, CA). Assay plates (96- well) were precoated with matrigel at a dilution of 1:400, and cells were seeded at a density of 60,000 cells/well (mT2R5), 75,000 cells/well
  • hTlRl/R3 expression was also initiated 48 hours prior to experiment.
  • Cell media was aspirated and 90 ⁇ l of pre-warmed HBSS or D-PBS was added to each well.
  • Cells were incubated for 45 minutes at 37°C, buffer was aspirated and 90 ⁇ l of pre-warmed agonist solutions in HBSS or D-PBS containing 50 ⁇ M rolipram and 0.7 to 5 ⁇ M forskolin was added to each of the corresponding wells. Plates were incubated for 15 minutes at 37°C.
  • Agonists were aspirated and stimulation was terminated with addition of 60 ⁇ l of lysis buffer into each well.
  • cAMP levels were then determined as described by the kit instructions. An independent cAMP standard curve was performed on each 96- well plates used. Chemiluminescent signals were detected using a TopCount- NXT (PerkinElmer, Wellesley, MA) set at a read-time of 2 seconds/well.
  • a flavor acceptance study is conducted using a test composition comprising a TIR or T2R modulator identified according to the foregoing examples.
  • a control composition lacking the TIR or T2R modulator, but which is otherwise substantially similar or identical to the test composition, is also used.
  • the study employs a two-way crossover design, with all subjects evaluating both compositions, which are administered in one or more same amounts or doses.
  • the test and control compositions are evaluated on a single study day.
  • the sequence for administering the test and control compositions is randomized among subjects. All enrolled subjects complete all aspects of the study protocol.
  • a clone stably expressing mT2R5 shows robust induction of ERKl/2 phosphorylation upon exposure to cycloheximide (Figure 1A).
  • Activation of ERKl/2 by cycloheximide in mT2R5-expressing cells peaks at 3-5 minutes post-stimulation ( Figure IB).
  • Other bitter substances including quinine and denatonium benzoate, sweeteners such as saccharin or sucrose and MSG do not induce ERKl/2 activation in mT2R5-expressing cells ( Figure 1A).
  • stimulation of rT2R9, the rat receptor orthologue of mT2R5 (85) Bufe et al, Nat. Genet.
  • Cycloheximide activates ERKl/2 in a dose-dependent fashion in mT2R5-expressing cells with an ECso of 1.1 +/-0.4 ⁇ M (mean +/- SD of three independent determinations) ( Figure ID). Saccharin and sucrose also induce ERKl/2 activation in a dose-dependent fashion in hTlR2/R3 -expressing cells ( Figure 3A and 3B).
  • Sweeteners such as aspartame, cyclamate, saccharin and monellin decrease forskolin-induced cAMP accumulation levels by 55%, 40%, 55% and 64% respectively and in a PTX-sensitive fashion (Figure 5A).
  • Fructose and sucrose do not inhibit cAMP accumulation in hTlR2/R3-expressing cells, on the contrary; fructose apparently increase cAMP levels (Figure 5A).
  • the lack of apparent effect of fructose and sucrose in the inhibition assay can be explained by the fact that these two sweeteners consistently increase cAMP levels in HEK293 cells not expressing the sweet receptor (Figure 5B).
  • Cyclamate (Figure 5C), aspartame (Figure 5D) and saccharin (Figure 5E) inhibit cAMP accumulation in a dose-dependent fashion with ECsos of 1.2 +/- 0.7mM, 350 +/- 60 ⁇ M and 61 +/- 33 ⁇ M respectively (Figure 5C) (mean +/- SD of three independent determinations).
  • hTlRl/hTlR3 umami taste receptor line exhibits a very high basal cAMP level relative to our mT2R5 and hTlR2/hTlR3 lines (mT2R5 line: 2.8 +/- 1.9 pmol/well, T2R2/R3 line: 4.5 +/- 1.9 pmol/well, hTlRl/hTlR3 line: 180 +/- 30 pmol/well).
  • Sweet and bitter receptors do not couple to Gs-stimulation in HEK293 cells.
  • Current models suggest that the sweet receptor may couple to GS to increase cAMP levels in TRCs (9, 10) (Gilbertson et al (2000); Margolskee (2002)).
  • ERKl/2 activation and inhibition of cAMP accumulation point to a direct coupling to Gi proteins ( Figure 2, 8 and 5).
  • this receptor could have dual properties, coupling to both Gi and G s . Therefore, we sought to determine if we could detect an agonist-induce increase in cAMP levels in the hTlR2/R3 sweet taste receptor line. Under these experimental conditions (i.e.
  • cAMP levels remain unchanged after stimulation with aspartame, cyclamate, saccharin and monellin (Figure 7A).
  • a ⁇ -adrenergic receptor ( ⁇ 2AR) agonist induces a 100% increase of cAMP accumulation in hTlR2/hTlR3-expressing cells indicating that a functional receptor/G s interection can be detected under these experimental conditions.
  • the sweeteners do not induce an increase of CAMP levels even after inhibiting functional coupling to Gi With PTX ( Figure 7B).
  • cAMP is a universal second messenger used by a plethora of cell surface receptors to relay signals from the extracellular milieu to the intracellular signaling machinery such as protein kinases, transcription factors and ion channels (89, 90, 92) (Morris and Malbon (1999); Chin et al (2002); Robinson- White and Stratakis, Ann NY Acad. Sci.
  • GPCRs activation of Gas and G ⁇ i respectively increase and decrease intracellular cAMP levels (Hanoune and Defer, Annu Rev. Pharmacol Toxical 42: 145-174 (2001) (39)) (Hansom and Defr (2001)).
  • the GTP-bound form of G ⁇ s directly interacts and activates the 9 types of membrane-bound adenylyl cyclase (AC) known (93).
  • AC membrane-bound adenylyl cyclase
  • GTP-bound form of G ⁇ i can directly interact and inhibit up to 6 different types of AC (39).
  • ERKl/2 is activated by G q , G s and Gi-coupled GPCRs (Liebmann et al (1996); Pierce et al., Oncogene 20(13): 1532-1539 (2001); Gutkind, J.S., J. Biol Chem 273(4): 1839-42 (1998) (91, 94, 95)) and, depending on the cellular context, several signaling pathways can be triggered to activate ERKl/2. Specifically, it is thought that Gi-coupled GPCRs activate ERKl/2 mainly via the free (activated) G ⁇ subunits (Crespo et al. Nature 369: 418-20 (1994); Faure et al, J. Biol Chem.
  • a rodent bitter receptor mT2R5, the human sweet taste receptor, hTlR2/hTlR3, and the human umami taste receptor, hTlRl/R3, couples to the activation of ERKl/2 and the inhibition of cAMP accumulation in HEK293 cells.
  • the bitter substance cycloheximide, the sweeteners saccharin, sucrose, cyclamate, D-tryptophan and the flavory amino acid MSG activate ERKl/2 exclusively in cells expressing their respective receptors.
  • cycloheximide on mT2R5, saccharin and sucrose on hTlR2/R3 and MSG on hTlRl/R3 reach saturation at higher concentrations and their potency at activating ERKl/2 is similar to the ones reported for the Gis-induced calcium mobilization in HEK293 (80, 14) (Chandrashekar et al (2000); Li et al., (2002)).
  • cycloheximide, artificial sweeteners, a sweet protein as well as MSG decrease cAMP levels exclusively in cells expressing their respective taste receptors.
  • ⁇ -subunits of the Gi family including G ⁇ u-i, G ⁇ -2, G ⁇ u-a, G ⁇ io-i, G ⁇ io-2, ⁇ -transducin and ⁇ -gustducin contain a conserved carboxyl-terminal cystein residue that is a site for modification by PTX, a 5'-diphosphate-ribosyltransferase isolated from Bortadella pertussis (101) (Fields et al. Biochem J. 321(Pl-3): 561- 71 (1997)).
  • PTX specifically and irreversibly modifies these G-protein subunits in vivo with attachment of an ADP-ribose moiety and, as a result, this covalent modification physically uncouples the G-protein from activation by GPCRs (101) (Fields et al. (1997)).
  • GPCRs (101)
  • incubation of cells with PTX abolishes the activation of ERKl/2 by the bitter, sweet and umami taste receptors indicating that one or more members of the Gi family functionally link the taste receptors to this signaling pathway in HEK293 cells. It is very likely that a-subunits of G ⁇ n-3 subfamily are involved since expression of G ⁇ -2 is restricted to the brain (Offermanns, S.
  • cAMP could have more of a modulator role, controlling intensity and/or the duration of taste sensation.
  • cAMP response element-binding protein (CREB) and phosphorylated- CREB have been recently localized in TRCs (55), suggesting that gene expression regulation can be potentially controlled, at least in part, by the level of cAMP in TRCs.
  • sweeteners such as saccharin and sucrose were shown to increase cAMP levels in rat taste epithelium (Striem et al., 1989 (78)), in mouse fungiform taste buds (Nakashima and Ninomiya, Cell Physiol. Biochem. 9(2):90-98 (1999) (112)) and in pig circumvallate papillae (77, Nairn et al., 1991).
  • these observations have led to the suggestion that the sweet receptor couples to Gs in TRCs (9, 10). In our hands, however, the sweet receptor clearly couples to a reduction of intracellular cAMP levels and activation of ERKl/2 through the direct functional coupling with Gi.
  • TRCs TRCs
  • PLC ⁇ 2 phospholipase C- ⁇ 2
  • IP3R-III type-Ill inositol trisphosphate receptor
  • TRPM5 transient receptor potential

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Abstract

La présente invention est en partie caractérisée par la découverte que des protéines G autres que la Gα15 réalise un couplage avec des récepteurs gustatifs T1R ou T2R, notamment les protéines GI, telles que la Gαi. En relation avec cette découverte, l'invention a trait à des procédés de dosage à base de cellules pour l'identification de composés modulateurs de l'activité spécifique des récepteurs gustatifs T1R ou T2R ou qui sont modulateurs de l'effet d'autres modulateurs de T1R ou T2R sur l'activité de T1R ou T2R. Ces procédés de dosage effectuent, de préférence, la détection de l'effet d'un composé modulateur présumé de T1R ou T2R sur une activation de MAPK, une accumulation d'AMPc, ou une activité d'adénylcyclase ou autre voie de signalisation régulé par des protéines Gi. Le niveau d'activation MAPK, d'accumulation AMPc ou d'adénylcyclase est, de préférence, déterminée par des procédés de dosage immunologique qui utilisent des ligands (anticorps monoclonaux ou polyclonaux) de liaison spécifique à un MAPK, AMPc, ou adénylcyclase activé (phosphorylé).
PCT/US2004/002987 2003-02-03 2004-02-03 Couplage fonctionnel de t1rs et de t2rs par des proteines gi, et dosages a base de cellules pour l'identification de modulateurs de t1r et t2r WO2004069191A2 (fr)

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WO2008119195A1 (fr) * 2007-03-30 2008-10-09 Givaudan Sa Procédé d'identification de modulateurs
WO2009149577A1 (fr) * 2008-06-13 2009-12-17 Givaudan Sa Procédés d’identification de modulateurs du récepteur de goût amer tas2r44
WO2011138455A1 (fr) * 2010-05-07 2011-11-10 Givaudan Sa Procédé pour identifier des modulateurs
EP3147363A2 (fr) 2015-09-26 2017-03-29 B.R.A.I.N. Ag Activation de gènes des récepteurs gustatives dans cellules mammifières á coup de crispr-cas-9
US10293024B2 (en) * 2013-08-15 2019-05-21 Blueberry Therapeutics Limited MAP kinase P38 binding compounds
WO2021050394A1 (fr) * 2019-09-09 2021-03-18 Firmenich Incorporated Bloqueurs du récepteur de goût amer et procédés pour leur identification
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WO2007127479A2 (fr) * 2006-04-28 2007-11-08 Redpoint Bio Corporation Dérivés d'imidazole substitués par triaryle et leurs utilisations dans l'inhibition du goût
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EP2742350B1 (fr) 2011-08-08 2019-10-30 The Coca-Cola Company Lignées cellulaires comprenant des récepteurs endogènes du goût, et leurs utilisations
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EP1865316A1 (fr) 2006-06-07 2007-12-12 Nutrinova Nutrition Specialties & Food Ingredients GmbH Procédé de criblage de composés qui modulent l'activité de gpcrs
US8067235B2 (en) 2006-06-07 2011-11-29 Nutrinova Nutrition Specialties & Food Ingredients Gmbh Optimized human T1R2 nucleic acid molecule
US8067236B2 (en) 2006-06-07 2011-11-29 Nutrinova Nutrition Specialties & Food Ingredients Gmbh Optimized human T1R3 nucleic acid molecule
US8597896B2 (en) 2007-03-30 2013-12-03 Givaudan S.A. Methods to identify modulators using TAS2R bitter taste receptor
WO2008119195A1 (fr) * 2007-03-30 2008-10-09 Givaudan Sa Procédé d'identification de modulateurs
US8187822B2 (en) 2007-03-30 2012-05-29 Givaudan S.A. Methods to identify modulators
WO2009149577A1 (fr) * 2008-06-13 2009-12-17 Givaudan Sa Procédés d’identification de modulateurs du récepteur de goût amer tas2r44
WO2011138455A1 (fr) * 2010-05-07 2011-11-10 Givaudan Sa Procédé pour identifier des modulateurs
US10293024B2 (en) * 2013-08-15 2019-05-21 Blueberry Therapeutics Limited MAP kinase P38 binding compounds
EP3147363A2 (fr) 2015-09-26 2017-03-29 B.R.A.I.N. Ag Activation de gènes des récepteurs gustatives dans cellules mammifières á coup de crispr-cas-9
WO2017050963A1 (fr) 2015-09-26 2017-03-30 B.R.A.I.N. Ag Activation de gènes de récepteurs de goûts dans des cellules de mammifères à l'aide de crispr-cas9
EP3147363A3 (fr) * 2015-09-26 2017-06-14 B.R.A.I.N. Ag Activation de gènes des récepteurs gustatives dans cellules mammifières á coup de crispr-cas-9
US11459582B2 (en) 2015-09-26 2022-10-04 B.R.A.I.N. Ag Activation of taste receptor genes in mammalian cells using CRISPR-Cas-9
WO2021050394A1 (fr) * 2019-09-09 2021-03-18 Firmenich Incorporated Bloqueurs du récepteur de goût amer et procédés pour leur identification
WO2023205647A1 (fr) * 2022-04-19 2023-10-26 Firmenich Incorporated Réduction du goût amer des principes actifs pharmaceutiques et dosages et procédés de criblage associés

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